JP3770333B2 - Recombinant DNA virus and method for producing the same - Google Patents

Description

【００２８】
▲４▼ カセットコスミドｐＡｄｅｘｌｐＣＡｗの構築
▲１▼で構築したプラスミドｐＣＭｗＣＨ３１をＨｉｎｄIII およびＣｌａＩで同時消化し、Ｋｌｅｎｏｗ酵素により平滑化し、5'末端リン酸化を施したＰｍｅＩリンカーとのライゲーションを行う。リガーゼを熱失活させた後、Ｐｓｐ１４０６Ｉで消化する。この切断はリンカーが複数個挿入されたものからＰｓｐ１４０６Ｉ断片を切り出し、リンカーがＤＮＡ断片の両端にそれぞれ１個連結した構造の断片を得るために行う。このあと、反応液をアガロースゲル電気泳動に供し、２．３ｋｂのＤＮＡ断片を含むゲルを切り出し、電気泳動によりゲルからＤＮＡ断片を回収する。次に、ｐＡｄｅｘｌｃｗをＣｌａＩで切断した後、生じた小断片をＳｐｕｎ ｃｏｌｕｍｎ（Ｐｈａｒｍａｃｉａ製）により除去した後のＤＮＡ断片と前述の２．３ｋｂのＤＮＡ断片をＴ４ＤＮＡリガーゼでライゲーションさせる。リガーゼを熱失活させた後、ＣｌａＩを添加し、セルフアニーリングにより生じた環状コスミドを切断する。ついで、イン・ビトロ・パッケージングに用いる。
更に、各コロニーから調製したコスミドＤＮＡの構造をＢａｍＨＩおよびＸｈｏＩ同時消化により確認する。目的とする方向に挿入された場合４８３ｂｐと４．８ｋｂ、逆方向に挿入された場合、２．７及び２．５ｋｂの断片を生じる。この確認により目的とするカセットコスミドｐＡｄｅｘｌｐＣＡｗを取得する。
【００２９】
▲５▼ カセットコスミドｐＡｄｅｘｌＣＡｗｔ（細胞工学、Vol.１３、No.8、Ｐ７５９）の構築
ＳｗａＩで切断した後エタノール沈澱により回収したｐＡｄｅｘｌｐＣＡｗを、5'末端リン酸化を施した下記の合成リンカー（２）（配列番号：３）（ＣｌａＩ、ＸｂａＩ、ＳｐｅＩ、ＰａｃＩ、ＳｗａＩ、ＣｌａＩ部位を含む）を混合し、ＡＴＰ、Ｔ４ＤＮＡリガーゼを含む反応液中で一晩結合させる。リガーゼを熱失活させた後、ＰａｃＩ（２０ｕｎｉｔ）を添加し、反応させる。この切断はリンカーが複数個挿入されたものからＰａｃＩ断片を切り出し、リンカーが１個のみ挿入された構造のコスミドを得るために行う。次に、反応液をＳｐｕｎ ｃｏｌｕｍｎ（Ｐｈａｒｍａｃｉａ製）にかけ、リンカー由来の小断片を除去した後、Ｔ４ＤＮＡリガーゼを含む反応液中で一晩結合させ、セルフアニーリングによる環状化を行う。リガーゼを熱失活させた後、イン・ビトロ・パッケージングに用いる。次に、各コロニーから調製したコスミドＤＮＡの構造をＸｂａＩおよびＸｈｏＩ同時消化により確認する。目的とする方向に挿入された場合５５２ｂｐ、逆方向に挿入された場合、５６８ｂｐの断片を生じる。これを確認することにより目的とするカセットコスミドｐＡｄｅｘｌＣＡｗｔ（図２）を取得する。
合成リンカーの構造

【００３０】
（ｂ）（ｌｏｘＰ挿入コスミドの構築その１）
ｐＡｄｅｘ２Ｌ３ＬＣＡｗｔの作製
▲１▼ ｐＡ６０Ｘ９９Ｘの作製
ｐＡｄｅｘｌＣＡｗｔをＢａｍＨＩで切断した後、熱処理によりＢａｍＨＩを失活させる。次にＴ４ＤＮＡリガーゼにより一晩結合させる。次いでこの反応混液を用いて大腸菌ＤＨ５α（ＧＩＢＣＯ ＢＲＬ製）を形質転換し、得られた形質転換体からプラスミドＤＮＡを調製し、目的とするプラスミドｐＡ６０Ｘ９９Ｘを得る。
【００３１】
▲２▼ ｐＡ６０Ｘ９９の作製（アデノウイルス以外のＸｂａＩ部位の除去）
ｐＡ６０Ｘ９９ＸをＸｂａＩ処理し、反応混液をアガロースゲル電気泳動し、２ヶ所のＸｂａＩ部位のうち１ヶ所のみで切断された２３．８ｋｂのＤＮＡ断片を含むゲルを切り出し、電気泳動によりゲルからＤＮＡ断片を回収する。次に、この断片をＫｌｅｎｏｗ酵素（宝酒造製）で両末端を平滑化し、Ｔ４ＤＮＡリガーゼで一晩結合させる。次いでこの反応混液を用いて大腸菌ＤＨ５αを形質転換し、得られた形質転換体からプラスミドＤＮＡを調製する。これらのプラスミドＤＮＡをＢｓｒＧＩおよびＸｂａＩで同時消化し、６．２ｋｂの断片すなわちプラスミドｐＡ６０Ｘ９９（図３）を得る。
【００３２】
▲３▼ ｐＡ２Ｌ６０Ｘ９９の作製（ＢｓｒＧＩ部位へのｌｏｘＰの挿入）
ｐＵＬＬ２ｒをＸｈｏｌで切断した後、Ｋｌｅｎｏｗ酵素（宝酒造製）で両末端を平滑化する。その後フェノール：クロロホルム（１：１）処理を施した後、エタノール沈澱する。沈澱物を遠心分離により取得し、ＴＥ６０μｌに溶解する。これと５’末端リン酸化ＫｐｎＩリンカー（宝酒造製）、ＡＴＰおよびＴ４ＤＮＡリガーゼを含むリガーゼ反応液（最終容量５０μｌ）中で一晩結合させる。次に、Ａｓｐ７１８（ベーリンガー製）で消化する。反応混液をアガロースゲル電気泳動し、ｌｏｘＰを含む６４ｂｐのＤＮＡ断片を含むゲルを切り出し、電気泳動によりゲルからＤＮＡ断片を回収する。
【００３３】
なお、上記のｐＵＬＬ２ｒは以下のようにして調製する。ｐＵＣ１１９（宝酒造製）を制限酵素Ｅｃｌ１３６IIで切断し、アルカリホスファターゼ処理を施した後、末端にＭｌｕＩ部位およびＸｈｏＩ部位を有しこれが連結するとＮｒｕＩ部位を生じるように設計されているｌｏｘＰ配列を含む下記の合成ＤＮＡ断片（配列番号：４）とのligation反応を行い該合成ＤＮＡ断片が２つ挿入されたプラスミドｐＵＬＬ２ｒを得る。

（下線部分の配列がｌｏｘＰ部位である。）
【００３４】
一方、プラスミドｐＡ６０Ｘ９９（１０μｇ）をＢｓｒＧＩ（５０ｕｎｉｔ）を含む反応系５０μｌ中で切断した後、反応混液をアガロースゲル電気泳動し、２３．８ｋｂのＤＮＡ断片を含むゲルを切り出し、電気泳動によりゲルからＤＮＡ断片を回収する。このＤＮＡ断片と前述のｌｏｘＰを含む６４ｂｐのＤＮＡ断片、ＡＴＰおよびＴ４ＤＮＡリガーゼを含むリガーゼ反応液中で一晩結合させる。これに滅菌水、ＢｓｒＧＩ反応ｂｕｆｆｅｒを加え、７０℃で１０分間インキュベートすることによりリガーゼを熱失活させた後、ＢｓｒＧＩで処理してセルフアニーリングにより生じた環状のｐＡ６０Ｘ９９を切断する。次いでこの反応混液１０μｌを用いて大腸菌ＤＨ５αを形質転換し、得られた形質転換体からプラスミドＤＮＡを調製する。
【００３５】
挿入されたｌｏｘＰの方向を確認するため、ＡｐａＩとＭｌｕＩの同時消化を行い、反応混液をアガロースゲル電気泳動する。目的とする方向に挿入された場合、３６６および２１９ｂｐ、逆方向に挿入された場合、３３４および２５１ｂｐの断片が生じる。また、ＮｒｕＩで消化した場合、目的とする方向に挿入された場合５７３ｂｐ、逆方向に挿入された場合、５３３ｂｐの断片が生じる。さらに、ＤｒａＩとＫｐｎＩで同時消化した場合、目的とする方向にｌｏｘＰが１つ挿入された場合３２０ｂｐ、２つ挿入された場合、３８４ｂｐの断片が生じる。これら３種の条件をすべて満たす、すなわち、目的とする方向にｌｏｘＰが１つ挿入された目的のプラスミドｐＡ２Ｌ６０Ｘ９９（図４）を取得する。
【００３６】
▲４▼ ｐＡ２Ｌ３Ｌ６０９９の作製（ＸｂａＩ部位へのｌｏｘＰの挿入）
ｐＵＬＬ２ｒをＸｈｏＩを含む反応系１００μｌ中で切断した後、Ｋｌｅｎｏｗ酵素（宝酒造製）で両末端を平滑化する。ついで、フェノール：クロロホルム（１：１）処理を施した後、エタノール沈澱する。沈澱物を遠心分離により取得し、ＴＥに溶解する。これと５’末端リン酸化ＳｐｅＩリンカー（宝酒造製）、ＡＴＰおよびＴ４ＤＮＡリガーゼを含む反応液中で一晩結合させる。さらに、ＳｐｅＩを加え消化した後、反応混液をアガロースゲル電気泳動し、ｌｏｘＰを含む６４ｂｐのＤＮＡ断片を含むゲルを切り出し、電気泳動によりゲルからＤＮＡ断片を回収する。
【００３７】
一方、ｐＡ２Ｌ６０Ｘ９９を、ＸｂａＩで切断した後、反応混液をアガロースゲル電気泳動し、２３．８ｋｂのＤＮＡ断片を含むゲルを切り出し、電気泳動によりゲルからＤＮＡを回収する。このＤＮＡ断片と前述のｌｏｘＰを含む６４ｂｐのＤＮＡ断片、ＡＴＰおよびＴ４ＤＮＡリガーゼを含む反応液中で一晩結合させる。リガーゼを熱失活させ、ついでこれをＸｂａＩで処理し、セルフアニーリングにより生じた環状のｐＡ２Ｌ６０Ｘ９９を切断する。この反応混液を用いて大腸菌ＤＨ５αを形質転換し、得られた形質転換体からプラスミドＤＮＡを調製する。
【００３８】
挿入されたｌｏｘＰの方向を確認するため、ＢｇｌIIとＭｌｕＩの同時消化を行い、反応混液をアガロースゲル電気泳動する。目的とする方向に挿入された場合、３６６および５０３ｂｐ、逆方向に挿入された場合、３９８および４７１ｂｐの断片が生じる。また、ＡｐａＩとＭｌｕＩで同時消化した場合、目的とする方向に挿入された場合６６０ｂｐ、逆方向に挿入された場合、６２８ｂｐの断片が生じる。ＥｃｏＮＩとＭｌｕＩで消化した場合、目的とする方向に挿入された場合３１１ｂｐ、逆方向に挿入された場合、３４３ｂｐの断片が生じる。さらに、ＨｐａＩとＳａｃＩで同時消化した場合、目的とする方向にｌｏｘＰが１つ挿入された場合３９７ｂｐ、２つ挿入された場合、４６１ｂｐの断片が生じる。これら４種の条件をすべて満たす、すなわち、目的とする方向にｌｏｘＰが１つ挿入された目的のプラスミドｐＡ２Ｌ３Ｌ６０９９（図５）を取得する。
【００３９】
▲５▼ ｐＡｄｅｘ２Ｌ３ＬＣＡｗｔの作製
ｐＡｄｅｘ１ＣＡｗｔを、Ｃｓｐ４５Ｉ（東洋紡製）で切断し、次いで、同反応液中でＢａｍＨＩ、さらにＥｃｏＲＩで切断した後、アガロースゲル電気泳動によるチェックを行う。２１ｋｂのＤＮＡ断片を含むゲルを切り出し、電気泳動によりゲルからＤＮＡ断片を回収する。なお、Ｃｓｐ４５１およびＥｃｏＲＩによる切断は２１ｋｂのＢａｍＨＩＤＮＡ断片を回収する際、他の断片が混入するのを防ぐためである。
【００４０】
ｐＡ２Ｌ３Ｌ６０９９をＢａｍＨＩで切断した後、フェノール：クロロホルム（１：１）処理を施し、水層をＴＥで平衡化したＳｅｐｈａｄｅｘＧ２５によりゲル濾過する。回収したＤＮＡ断片と前述の２１ｋｂのＤＮＡ断片、ＡＴＰおよびＴ４ＤＮＡリガーゼを含む反応液中で一晩結合させる。リガーゼを熱失活させた後、これをイン・ビトロ・パッケージングに用いる。
【００４１】
即ち、ラムダ・イン・ビトロ・パッケージングキットであるギガバックＸＬ（Ｓｔｒａｔａｇｅｎｅ製）を用い、残りは−８０℃に凍結する。ギガバックＸＬは４２ｋｂ以下のコスミドのパッケージ効率が低いのでインサートが入って大きくなったコスミドをある程度選択することができる。本発明では、１０個のコロニーを拾えば大半はインサートを含んでおり、目的のクローン（ウイルスゲノムが正しく連結されたクローン）を容易に得ることができる。コスミドの扱い方については、常法（斎藤 泉他、実験医学：７：183-187, 1989)に従って行う。
【００４２】
パッケージングされたコスミドを大腸菌ＤＨ５α（ＧｉｂｃｏＢＲＬ製）に感染させる。即ち、Ａｐ⁺ （アンピシリン添加）寒天プレートとＡｐ⁺ ＬＢ（ｐｏｏｌ）に各種の濃度で接種し、一晩培養する。ｐｏｏｌのｍｉｎｉｐｒｅｐＤＮＡを抽出・調製し、制限酵素ＤｒａＩ切断によりインサートがはいったものの割合を調べる。コロニーは丸ごと寒天ごと取りＡｐ⁺ ＬＢで一晩培養し、ｍｉｎｉｐｒｅｐＤＮＡを調製する。次に、各コロニーから調製したコスミドＤＮＡの構造をＤｒａＩ切断により確認する。目的とする方向に挿入された場合８９１ｂｐ、逆方向に挿入された場合１．４ｋｂの断片を生じる。これにより目的とするカセットコスミドｐＡｄｅｘ２Ｌ３ＬＣＡｗｔを取得する。
【００４３】
（ｃ）（ｌｏｘＰ挿入コスミドの構築その２）
ｐＡｄｅｘ２ＬＡ３ＬＣＡｗｔの作製
▲１▼ ｐＵＣＡ６０６５の作製
ｐＡ６０Ｘ９９をＢａｍＨＩおよびＰｓｔＩにより切断し、反応混液をアガロースゲル電気泳動し、ＢｓｒＧＩ部位を含む１．７ｋｂのＤＮＡ断片を含むゲルを切り出し、電気泳動によりゲルからＤＮＡ断片を回収する。同様の操作によりｐＵＣ１９をＢａｍＨＩおよびＰｓｔＩにより切断し、２．７ｋｂの断片を回収する。次に両断片をリガーゼ反応ｂｕｆｆｅｒ中に加え、さらにＡＴＰ、Ｔ４ＤＮＡリガーゼを加え、一晩結合させる。次いでこの反応混液を用いて大腸菌ＤＨ５αを形質転換し、得られた形質転換体からプラスミドＤＮＡを調製し、目的とするプラスミドｐＵＣＡ６０６５を得る。
【００４４】
▲２▼ ｐ２ＬＡ６０６５の作製
ｐＵＣＡ６０６５をＢａｍＨＩおよびＡｆｌIII で切断し、７８０ｂｐの断片を調製し、また、同プラスミドをＢａｍＨＩおよびＢｓｒＧＩで切断し、３．６ｋｂの断片を調製する。これら両断片とｌｏｘＰ配列を含む下記のリンカーＤＮＡ（配列番号：１１）を混合しリガーゼ反応ｂｕｆｆｅｒ中に加え、さらにＡＴＰ、Ｔ４ＤＮＡリガーゼを加え、一晩結合させる。次いでこの反応混液を用いて大腸菌ＤＨ５αを形質転換し、得られた形質転換体からプラスミドＤＮＡを調製し、リンカーＤＮＡが１つ挿入された、目的とするプラスミドｐ２ＬＡ６０６５を得る。

【００４５】
▲３▼ ｐＡ２ＬＡ３Ｌ６０９９の作製
ｐ２ＬＡ６０６５をＢａｍＨＩおよびＳｆｉＩ（あるいはＢｇｌＩ）により切断し、１．５ｋｂの断片を調製する。また、ｐＡ２Ｌ３Ｌ６０９９をＢａｍＨＩおよびＳｆｉＩにより切断し、約２２ｋｂの断片を調製する。次に両断片をリガーゼ反応ｂｕｆｆｅｒ中に加え、さらにＡＴＰ、Ｔ４ＤＮＡリガーゼを加え、一晩結合させる。次いでこの反応混液を用いて大腸菌ＤＨ５αを形質転換し、得られた形質転換体からプラスミドＤＮＡを調製し、目的とするプラスミドｐＡ２ＬＡ３Ｌ６０９９を得る。
【００４６】
▲４▼ ｐＡｄｅｘ２ＬＡ３ＬＣＡｗｔの作製
ｐＡｄｅｘ２Ｌ３ＬＣＡｗｔ作製の際の▲５▼と同様の操作により、ｐＡ２ＬＡ３Ｌ６０９９とｐＡｄｅｘｌＣＡｗｔからｐＡｄｅｘ２ＬＡ３ＬＣＡｗｔを作製する。
【００４７】
（ｄ）（ｌｏｘＰ挿入コスミドの構築その３）
ｐＡｄｅｘ２ＬＤ３ＬＣＡｗｔの作製
▲１▼ ｐＨＳＧＡ６０６５の作製
ｐＡ６０Ｘ９９をＢａｍＨＩおよびＰｓｔＩにより切断し、反応混液をアガロースゲル電気泳動し、ＢｓｒＧＩ部位を含む１．７ｋｂのＤＮＡ断片を含むゲルを切り出し、電気泳動によりゲルからＤＮＡ断片を回収する。ｐＨＳＧ２９９（宝酒造）をＢａｍＨＩおよびＰｓｔＩにより切断し、同様の操作により２．７ｋｂの断片を回収する。次に両断片をリガーゼ反応ｂｕｆｆｅｒ中に加え、さらにＡＴＰ、Ｔ４ＤＮＡリガーゼを加え、一晩結合させる。次いでこの反応混液を用いて大腸菌ＤＨ５αを形質転換し、得られた形質転換体からプラスミドＤＮＡを調製し、目的とするプラスミドｐＨＳＧＡ６０６５を得る。
【００４８】
▲２▼ ｐ２ＬＤ６０６５の作製
ｐＨＳＧＡ６０６５をＢｓｒＧＩおよびＤｒａＩで切断し４．４ｋｂの断片を調製し、これとｌｏｘＰ配列を含む下記のリンカーＤＮＡ（配列番号：１２）を混合し、リガーゼ反応ｂｕｆｆｅｒ中に加え、さらにＡＴＰ、Ｔ４ＤＮＡリガーゼを加え、一晩結合させる。次いでこの反応混液を用いて大腸菌ＤＨ５αを形質転換し、得られた形質転換体からプラスミドＤＮＡを調製し、リンカーＤＮＡが１つ挿入された目的とするプラスミドｐ２ＬＤ６０６５を得る。

【００４９】
▲３▼ ｐＡ２ＬＤ３Ｌ６０９９の作製
ｐ２ＬＤ６０６５をＢａｍＨＩおよびＳｆｉＩ（あるいはＢｇｌＩ）により切断し１．５ｋｂの断片を調製する。また、ｐＡ２Ｌ３Ｌ６０９９をＢａｍＨＩおよびＳｆｉＩにより切断し、約２２ｋｂの断片を調製する。次に両断片をリガーゼ反応ｂｕｆｆｅｒ中に加え、さらにＡＴＰ、Ｔ４ＤＮＡリガーゼを加え、一晩結合させる。次いでこの反応混液を用いて大腸菌ＤＨ５αを形質転換し、得られた形質転換体からプラスミドＤＮＡを調製し、目的とするプラスミドｐＡ２ＬＤ３Ｌ６０９９を得る。
【００５０】
▲４▼ ｐＡｄｅｘ２ＬＤ３ＬＣＡｗｔの作製
ｐＡｄｅｘ２Ｌ３ＬＣＡｗｔ作製の際の▲５▼と同様の操作により、ｐＡ２ＬＤ３Ｌ６０９９とｐＡｄｅｘｌＣＡｗｔからｐＡｄｅｘ２ＬＤ３ＬＣＡｗｔを作製する。
【００５１】
（ｅ）アデノウイルスＤＮＡ−末端蛋白複合体（Ａｄ５ ｄｌＸ ＤＮＡ−ＴＰＣおよびＡｄｅｘ１ＣＡＮＬａｃＺ ＤＮＡ−ＴＰＣ）の調製
▲１▼ アデノウイルスＤＮＡとしては、Ａｄ５ ｄｌＸ（I. Saito et al., J.Virology, vol.54, 711-719 (1985))またはＡｄｅｘ１ＣＡＮＬａｃＺを用いる。Ａｄ５ ｄｌＸをＨｅＬａ細胞（Ｒｏｕｘ １０本分）に、Ａｄｅｘ１ＣＡＮＬａｃＺを２９３細胞にそれぞれ感染させ、培養を行う。
即ち、Ａｄ５−ｄｌＸまたはＡｄｅｘ１ＣＡＮＬａｃＺのウイルス液（〜１０⁹ ＰＦＵ／ｍｌ）を０．２ｍｌ／Ｒｏｕｘ感染させ、３日後に、はがれた細胞を遠心分離により集める。アデノウイルス粒子のほとんどはメディウム中ではなく細胞の核内にいるので感染細胞からウイルスを精製できる利点がある。（以下の操作は非無菌的に行う。）
【００５２】
▲２▼ 得られた細胞をＴｒｉｓ−ＨＣｌ（ｐＨ８．０）に懸濁し、密封型ソニケーターを用いて細胞を破砕し、ウイルスを細胞内から放出させる。
▲３▼ 得られた破砕物を遠心分離により沈澱を除いた後、超遠心分離機 ＳＷ２８チューブに塩化セシウム溶液（比重１．４３）を入れ、その上に上清を重層し、クッション遠心による濃縮を行う。
▲４▼ 界面直下のウイルス層をＳＷ５０．１チューブに移す。界面直下のウイルス層は通常目視でき、ウイルス層とその下層の塩化セシウムを５ｍｌ採取する。同時にもう一本に塩化セシウム溶液（比重１．３４）を満たす。
これらを、３５ｋｒｐｍ、４℃で一晩超遠心にかけた。次いで、白いウイルスのバンドを分取し、既に勾配ができたチューブに乗せ替える。さらに、３５ｋｒｐｍ、４℃４時間以上超遠心にかける。
【００５３】
▲５▼ 白いウイルスのバンドを分取し、等量の８Ｍ塩酸グアニジンと室温で混合し、４Ｍ塩酸グアニジン飽和塩化セシウムを加えてＶＴｉ６５チューブに満たす。４Ｍ塩酸グアニジンにより、粒子蛋白は変性を受けて解離し、ＤＮＡ−ＴＰＣが放出される。
【００５４】
▲６▼ 上記のチューブを、５５ｋｒｐｍ、１５℃で一晩超遠心にかけ、０．２ｍｌずつ分画し、その１μｌずつを１μｇ／ｍｌのエチジウムブロミド水溶液２０μｌと混合し、蛍光染色することによりＤＮＡの有無を確認する。ＤＮＡを含む２〜３フラクションを集める。
▲７▼ ５００ｍｌのＴＥに一晩透析（２回）し、−８０℃に保存した。こうして得られたＡｄ５ｄｌＸ ＤＮＡ−ＴＰＣまたはＡｄｅｘ１ＣＡＮＬａｃＺ ＤＮＡ−ＴＰＣの量をＯＤ₂₆₀ から通常のＤＮＡと同様に算出する。
▲８▼ 得られたＡｄ５ｄｌＸ ＤＮＡ−ＴＰＣまたはＡｄｅｘ１ＣＡＮＬａｃＺＤＮＡ−ＴＰＣを、次のステップのｌｏｘＰ挿入組換えアデノウイルス作成のため、充分量のＡｇｅＩで２時間切断し、Ｓｅｐｈａｄｅｘ Ｇ２５カラムでゲル濾過後、−８０℃に保存する。
【００５５】
（ｆ）ｌｏｘＰ挿入組み換えアデノウイルスの作製
なお、ＮＬａｃＺは大腸菌ＬａｃＺ遺伝子のＮ末端にＳＶ４０の核移行シグナルを付加したものである。
▲１▼ １０％ＦＣＳ添加ＤＭＥで培養した２９３細胞の６ｃｍ、１０ｃｍシャーレ各１枚用意する。
▲２▼−１．（Ａｄ５ｄｌＸ２Ｌ３ＬまたはＡｄｅｘ２Ｌ３ＬＣＡＮＬａｃＺの作製）
発現ユニットを組み込んだｌｏｘＰを挿入したコスミドｐＡｄｅｘ２Ｌ３ＬＣＡｗｔ ＤＮＡの８μｇとＡｇｅＩで切断したＡｄ５ｄｌＸ ＤＮＡ−ＴＰＣまたはＡｇｅＩで切断したＡｄｅｘ１ＣＡＮＬａｃＺ ＤＮＡ−ＴＰＣの１μｇを混合し、セルフェクト（ファルマシア製）キットを用いて、６ｃｍシャーレ１枚にリン酸カルシウム法でトランスフェクションを行う。６ｃｍシャーレのメディウムの上から混合液を滴下し、培養を続ける。
一晩培養（約１６時間）し、午前中に培養液を交換し、夕方、コラーゲンコート９６穴３枚（原液・１０倍希釈・１００倍希釈）に、５％ＦＣＳ添加ＤＭＥを用い、各ウエル当たり０．１ｍｌでまき直す。細胞数が各プレートで大きく違わないように、希釈２枚分には１０ｃｍシャーレの２９３細胞を１／３ずつ混ぜて播く。
【００５６】
▲２▼−２．（Ａｄ５ｄｌＸ２ＬＡ３ＬまたはＡｄｅｘ２ＬＡ３ＬＣＡＮＬａｃＺの作製）
発現ユニットを組み込んだｌｏｘＰを挿入したコスミドｐＡｄｅｘ２ＬＡ３ＬＣＡｗｔ ＤＮＡの８μｇとＡｇｅＩで切断したＡｄ５ｄｌＸ ＤＮＡ−ＴＰＣまたはＡｇｅＩで切断したＡｄｅｘ１ＣＡＮＬａｃＺ ＤＮＡ−ＴＰＣの１μｇを混合し、セルフェクト（ファルマシア製）キットを用いて、６ｃｍシャーレ１枚にリン酸カルシウム法でトランスフェクションを行う。６ｃｍシャーレのメディウムの上から混合液を滴下し、培養を続ける。
一晩培養（約１６時間）し、午前中に培養液を交換し、夕方、コラーゲンコート９６穴３枚（原液・１０倍希釈・１００倍希釈）に、５％ＦＣＳ添加ＤＭＥを用い、各ウエル当たり０．１ｍｌでまき直す。細胞数が各プレートで大きく違わないように、希釈２枚分には１０ｃｍシャーレの２９３細胞を１／３ずつ混ぜて播く。
【００５７】
▲２▼−３．（Ａｄ５ｄｌＸ２ＬＤ３ＬまたはＡｄｅｘ２ＬＤ３ＬＣＡＮＬａｃＺの作製）
発現ユニットを組み込んだｌｏｘＰを挿入したコスミドｐＡｄｅｘ２ＬＤ３ＬＣＡｗｔ ＤＮＡの８μｇとＡｇｅＩで切断したＡｄ５ｄｌＸ ＤＮＡ−ＴＰＣまたはＡｇｅＩで切断したＡｄｅｘ１ＣＡＮＬａｃＺ ＤＮＡ−ＴＰＣの１μｇを混合し、セルフェクト（ファルマシア製）キットを用いて、６ｃｍシャーレ１枚にリン酸カルシウム法でトランスフェクションを行う。６ｃｍシャーレのメディウムの上から混合液を滴下し、培養を続ける。
一晩培養（約１６時間）し、午前中に培養液を交換し、夕方、コラーゲンコート９６穴３枚（原液・１０倍希釈・１００倍希釈）に、５％ＦＣＳ添加ＤＭＥを用い、各ウエル当たり０．１ｍｌでまき直す。細胞数が各プレートで大きく違わないように、希釈２枚分には１０ｃｍシャーレの２９３細胞を１／３ずつ混ぜて播く。
【００５８】
▲３▼ ３〜４日後と８〜１０日後に、各ウエルに５０μｌの１０％ＦＣＳ添加ＤＭＥを加える。２９３細胞がやせてきたら早めに加える。
ウイルスが増殖し細胞が死滅したウエルが７〜２０日の間に現れる。ウエルの細胞が完全に死滅するごとに滅菌パスツールピペットで培養液（死細胞ごと）を滅菌した１．５ｍｌチューブに無菌的に移して、ドライアイスで急凍して−８０℃に保存する。
▲４▼ １５〜２５日で判定は終了する。比較的遅く細胞が死んだウエルから回収した培養液チューブを約１０個選び、超音波破砕後、５０００ｒｐｍ１０分遠心して得られた上清を１次ウイルス液（first seed）として−８０℃に保存する。早めにウイルス増殖が起こったウエルは複数のウイルス株の混合感染の可能性が高いからである。
【００５９】
▲５▼ ２４穴プレートに２９３細胞を用意し、５％ＦＣＳ−ＤＭＥ（０．４ｍｌ／ウエル）と１次ウイルス液１０μｌをそれぞれ２ウエルずつ添加する。
▲６▼ 約３日で細胞が完全に死滅したら、１ウエルは１次ウイルス液作製と同様に超音波破砕と遠心分離で上清を得、これを２次ウイルス液(second seed) として−８０℃に保存する。他の１ウエルの死滅した細胞を５０００ｒｐｍで５分間遠心し、上清を捨てて細胞だけを−８０℃に保存する（セルパック）。１０種類のウイルス株のセルパックが集まったら以下の方法で感染細胞の全ＤＮＡを抽出する。セルパックには、４００μｌのcell DNA用TNE (50mM Tris-HCl pH7.5, 100mM NaCl, 10mM EDTA)、４μｌのproteinaseK (10mg/ml) および４μｌの10%SDSを加える。
【００６０】
▲７▼ ５０℃で１時間処理した後、フェノール・クロロホルム抽出２回、クロロホルム抽出２回、ついでエタノール沈澱により得られた核酸をＲＮａｓｅを２０μｇ／ｍｌ含む５０μｌのＴＥに溶かす。その１５μｌを発現ユニットを切断する酵素の中で認識配列にＣＧを含む酵素であるＸｈｏＩで切断し、発現コスミドカセットのＸｈｏＩ切断と共に、１５ｃｍ位の長さのアガロースゲルで一晩電気泳動を行い、パターンを比較する。ＸｈｏＩは挿入したｌｏｘＰ配列内に認識部位があるので、ｌｏｘＰが２個挿入された切断パターンを示すクローンを選択する。説明できないバンドが薄く見えるクローンは、欠失のあるウイルスとの混合の可能性があるので廃棄する。
【００６１】
ここで得られるｌｏｘＰ挿入組換えアデノウイルスＡｄ５ｄｌＸ２Ｌ３Ｌ、Ａｄ５ｄｌＸ２ＬＡ３Ｌ、Ａｄ５ｄｌＸ２ＬＤ３Ｌ等と、目的の外来遺伝子発現ユニットを含むコスミドを用いて、公知の組換えアデノウイルス作製方法、例えばＣＯＳ−ＴＰＣ法（「実験医学別冊」バイオマニュアルシリーズ４、遺伝子導入と発現・解析法、４３〜５８頁）に従って、目的の外来遺伝子発現ユニットとｌｏｘＰが挿入された組換えアデノウイルスを作製することができる。
【００６２】
（ｇ）Ｅ２Ａ遺伝子欠損アデノウイルスの作製と構造確認
組み換えアデノウイルスＡｄｅｘ２Ｌ３ＬＣＡＮＬａｃＺおよびＡｄｅｘ１ＣＡＮＣｒｅをそれぞれｍｏｉ＝１０および３で２９３細胞に感染させ、培養を行う。４日目に細胞を回収し、前述の方法によりＤＮＡを調製する。ｌｏｘＰで挟まれた領域（Ｅ２Ａ遺伝子を含む）が切り出された構造を有するＡｄｅｘｄｌ２３ＣＡＮＬａｃＺの生成を２つの方法で確認する。
なお、Ａｄｅｘ２ＬＡ３ＬＣＡＮＬａｃＺおよびＡｄｅｘ２ＬＤ３ＬＣＡＮＬａｃＺに関しても同様の操作により構造が確認できる。
【００６３】
１．ＳｍａＩ消化
ＳｍａＩ消化の後、ゲル電気泳動した結果、ｌｏｘＰで挟まれた領域が切り出されて生ずる４．７ｋｂの断片が認められる。このバンドとＡｄｅｘ２Ｌ３ＬＣＡＮＬａｃＺ、Ａｄｅｘ１ＣＡＮＣｒｅ、およびＡｄｅｘｄｌ２３ＣＡＮＬａｃＺにおいて共通して見られる４．４５ｋｂのバンドの濃さの比較から、回収した組換えアデノウイルス中の約何％がＡｄｅｘｄｌ２３ＣＡＮＬａｃＺであるかが分かる。
【００６４】
２．ＰＣＲによる確認
調製したＤＮＡ０．１ｎｇをテンプレートとし、通常の条件でＰＣＲを行い、生成物をアガロースゲル電気泳動により分析する。用いるプライマーは、下記に示すオリゴヌクレオチド（１）（配列番号：５）、オリゴヌクレオチド（２）（配列番号：６）、オリゴヌクレオチド（３）（配列番号：７）およびオリゴヌクレオチド（４）（配列番号：８）が好ましい。

ＰＣＲ反応液組成は、１０ｍＭのＴｒｉｓ・ＨＣｌ（ｐＨ８．３）中、５０ｍＭのＫＣｌ、１．５ｍＭのＭｇＣｌ₂ 、０．２ｍＭのｄＮＴＰ mixture および各０．２μＭのプライマー、０．１ｎｇのテンプレートＤＮＡおよび０．５ｕｎｉｔのＴａｑ ポリメラーゼを含むものが好ましい。
ＰＣＲ反応条件としては、二本鎖解離を９５℃で１．５分、アニーリングを６４℃で１．０分、伸長反応を７０℃で１．０分、反応サイクル３０回、が好ましい１例である。
プライマーとして（１）と（４）を用いた場合、３９３ｂｐと推定されるバンドが検出され、Ｅ２Ａ遺伝子が欠失したＡｄｅｘｄｌ２３ＣＡＮＬａｃＺの存在が明らかとなり、また、（２）と（３）を用いた場合、２２１ｂｐと推定されるバンドが検出され、Ｃｒｅ遺伝子産物により切り出された環状のＥ２Ａ遺伝子の存在も裏付けられ、Ａｄｅｘ２Ｌ３ＬＣＡＮＬａｃＺからｌｏｘＰではさまれた領域（Ｅ２Ａ遺伝子を含む）が切り出されたＡｄｅｘｄｌ２３ＣＡＮＬａｃＺの生成が明らかになる（図６）。
【００６５】
（２） 次に、プロモーター、リコンビナーゼ遺伝子およびポリＡ配列を有する組換えアデノウイルスベクターの製造方法について説明する。
以下に、リコンビナーゼ遺伝子としてリコンビナーゼＣｒｅ遺伝子を使用した場合について述べるが、他のリコンビナーゼ遺伝子の場合もほぼ同様である。
【００６６】
▲１▼ ＰＣＲ法で調製したリコンビナーゼＣｒｅ遺伝子およびプラスミドｐＵＣ１９（宝酒造製）とをそれぞれ制限酵素ＰｓｔＩ（宝酒造製）およびＸｂａＩ（宝酒造製）で同時消化したのち混合・ライゲーションし、リコンビナーゼＣｒｅ遺伝子が組み込まれたプラスミドｐＵＣＣｒｅを得る。
▲２▼ ＣＡＧプロモーターを含むカセットコスミドｐＡｄｅｘ１ＣＡｗｔを制限酵素ＳｗａＩ（Boehringer製）で処理したものと、ｐＵＣＣｒｅを制限酵素ＰｓｔＩ（宝酒造製）およびＸｂａＩ（宝酒造製）で同時消化したのちＫｌｅｎｏｗ酵素（宝酒造製）で両端を平滑化したものとを混合する。ついでカセットコスミドを沈澱させ、Ｔ４ ＤＮＡリガーゼで結合させ、リコンビナーゼＣｒｅ遺伝子を組み込んだカセットコスミドを得る。
【００６７】
ＣＡＧプロモーター以外のプロモーターを使用する場合は、まず、アデノウイルスゲノム（３６ｋｂ）の全長のうち、複製に不要なＥ３領域（１．９ｋｂ）とＥ１Ａ・Ｅ１Ｂ領域（２．９ｋｂ）を欠失させた約３１ｋｂのゲノムＤＮＡをもつカセットコスミドを作成し、他方、使用しようとするプロモーター、リコンビナーゼＣｒｅ遺伝子およびポリＡ配列を含むプラスミドを作製し、適当な制限酵素で処理してアデノウイルスゲノムのＥ１Ａ・Ｅ１Ｂ欠失部位にリコンビナーゼＣｒｅ遺伝子発現ユニットを組み込んだカセットコスミドを得る。
▲３▼ 次に、得られたカセットコスミドを、ラムダ・インビトロ・パッケージングキットであるギガパックＸＬ（Ｓｔｒａｔａｇｅｎｅ製）を用いて、インビトロ・パッケージングを行う。
【００６８】
▲４▼ 一方、アデノウイルスＤＮＡ−末端蛋白複合体（Ａｄ５ｄｌＸ ＤＮＡ−ＴＰＣ）を調製する。アデノウイルスＤＮＡとしては、Ａｄ５ｄｌＸ（I. Saito et al., J.Virology, vol.54, 711-719 (1985) ）を用い、Ａｄ５ｄｌＸをＨｅＬａ細胞（Ｒｏｕｘ瓶１０本分）に感染させ、培養を行う。ウイルス粒子を回収し、塩酸グアニジン処理・超遠心によりＤＮＡ−ＴＰＣを分離・回収する。
こうして得られたＡｄ５ｄｌＸ ＤＮＡ−ＴＰＣを次のステップの組換えアデノウイルス作製のため充分量のＥｃｏＴ２２Ｉで処理する。
【００６９】
▲５▼ 最後のステップとして、リコンビナーゼＣｒｅ遺伝子を組み込んだカセットコスミドとＥｃｏＴ２２Ｉで処理したＡｄ５ｄｌＸ ＤＮＡ−ＴＰＣを混合し、セルフェクト（ファルマシア製）キットを用いてリン酸カルシウム法でトランスフェクションを行う。ウイルスの増殖のため細胞が死滅したものからウイルス液を回収しプロモーター、リコンビナーゼ遺伝子およびポリＡ配列を有する組換えアデノウイルスベクターを得る。
【００７０】
本発明の方法により上記のようにして得られる、目的の外来遺伝子発現ユニットを有し、Ｅ２Ａ遺伝子の機能が完全に欠失した本発明の組換えアデノウイルスの高力価ウイルス溶液は、適宜希釈して局所注入（中枢神経系・門脈など）、経口（腸溶剤を用いる）投与、経気道投与、経皮投与等の投与方法により共感染させ、遺伝病を含む各種疾患の治療に用いることができる。
【００７１】
【実施例】
以下、実施例、参考例により本発明をさらに詳しく説明するが、本発明はこれらの実施例等によりなんら限定されるものではない。
なお、実施例中のファージ、プラスミド、ＤＮＡ、各種酵素、大腸菌、培養細胞などを取り扱う諸操作は、特に断らない限り、「Molecular Cloning, A Laboratory Manual. T. Maniatis ら編、第２版（１９８9 ）、Cold Spring Harbor Laboratory 」に記載の方法に準じて行った。また、ＤＮＡ制限酵素および修飾酵素は、宝酒造、Ｎｅｗ Ｅｎｇｌａｎｄ Ｂｉｏｌａｂｓ（ＮＥＢ）社、Ｓｔｒａｔａｇｅｎｅ社又はＢｏｅｈｒｉｎｇｅｒ社から購入し、製造者指示書に従って使用した。
【００７２】
実施例１（ｐＡｄｅｘ１ＣＡｗｔの構築）
▲１▼ ＣＡＧプロモーターを含むプラスミドｐＣＭｗＣＨ３１の構築
ＣＡＧプロモーターを含むプラスミドｐＣＡＧＧＳ（Niwaら、Gene、108 、193-200(1990) ）をＥｃｏＲＩで切断した後、Ｋｌｅｎｏｗ酵素により平滑化し、ＳｗａＩリンカーとのリガーゼ反応を行った。次に、得られたプラスミドをＳａｌＩで切断した後、Ｋｌｅｎｏｗ酵素により平滑化し、ＣｌａＩリンカーとのリガーゼ反応を行った。さらに、得られたプラスミドをＰｓｔＩで切断した後、Ｋｌｅｎｏｗ酵素により平滑化し、ＸｈｏＩリンカーとのリガーゼ反応を行った。元の制限酵素部位の消失および各リンカーの挿入は各制限酵素切断の後、アガロースゲル電気泳動により確認した。
【００７３】
▲２▼ ｐＡｄｅｘ１ｃの構築
（ｉ）Ｅ１遺伝子領域を欠失したアデノウイルスゲノム左側末端の１７％を含むプラスミド（ｐＵＡＦ０−１７Ｄ）の調製
５型アデノウイルスＤＮＡをＳ１処理して平滑末端とし、その平滑末端にＢａｍＨリンカーを結合させ、その後ＨｉｎｄIII 消化し、目的のフラグメント（２．８ｋｂ、アデノウイルスゲノムの左側末端の８％に当たる）をアガロースゲル電気泳動で分離・回収し、ＢａｍＨＩ／ＨｉｎｄIII 消化したｐＵＣ１９のＢａｍＨＩ／ＨｉｎｄIII 部位へ挿入した。得られた目的のプラスミドをｐＵＡＦ０−８と名づけた。
【００７４】
（ii）アデノウイルスＤＮＡをＨｉｎｄIII 消化し、アガロースゲル電気泳動で分離し、目的の３．４ｋｂのフラグメント（アデノウイルスゲノムの左側末端の８−１７％に当たる）をゲルから回収し、ｐＵＣ１９のＨｉｎｄIII 部位へ挿入した（ｐＵＡＦ８−１７と命名）。
ｐＵＡＦ０−８の塩基番号（ここでいう塩基番号はアデノウイルスＤＮＡ由来）４５４番目のＰｖｕII部位をＣｌａＩリンカーを用いてＣｌａＩ部位に変換した。そして、このプラスミドをＢａｍＨＩ／ＣｌａＩ消化し、４５４ｂｐのＢａｍＨＩ−ＣｌａＩフラグメントをアガロースゲル電気泳動で回収した。
ｐＵＡＦ８−１７の塩基番号３３２８番目のＢｇｌII部位をＣｌａＩリンカーを用いてＣｌａＩ部位に変換した。そしてこのプラスミドをＨｉｎｄIII ／ＣｌａＩ消化し、２．９ｋｂのＨｉｎｄIII −ＣｌａＩフラグメントをアガロースゲル電気泳動で回収した。
ｐＵＡＦ０−８由来の４５４ｂｐのＢａｍＨＩ−ＣｌａＩフラグメントと、ｐＵＡＦ８−１７由来の２．９ｋｂのＨｉｎｄIII −ＣｌａＩフラグメントをつなぎ、ｐＵＣ１９のＢａｍＨＩ／ＨｉｎｄIII 部位へ挿入した。得られたプラスミドをｐＵＡＦ０−１７Ｄと命名した。このプラスミドはＥ１遺伝子領域を欠失したアデノウイルスゲノム左側末端の１７％を含む。
【００７５】
（iii)アデノウイルスゲノムのＢｓｔ１１０７−ＥｃｏＲＩフラグメント（２１．６ｋｂ）の調製
５型アデノウイルスＤＮＡをＢｓｔ１１０７とＥｃｏＲＩで消化し、アガロースゲル電気泳動で分離した後、目的の２１．６ｋｂのフラグメントを回収した。
【００７６】
（iv）アデノウイルスゲノムのＥｃｏＲＩ−ＳａｌＩフラグメント（６．５ｋｂ）の調製
ｐＸ２Ｓ（I. Saito et. al., J. of Virology, vol. 54, p711-719, 1985)のＳａｌＩ部位をＳｗａＩリンカー（メーカー名）を用いてＳｗａＩ部位へ変換しｐＸ２Ｗを得た。ｐＸ２ＷをＥｃｏＲＩとＳｗａＩで消化し、アガロースゲル電気泳動で分離した後、目的の６．５ｋｂのフラグメントを回収した。
【００７７】
（ｖ）シャロミド（ｃｈｄＲＢＲ７−１１）の調製
charomid９−１１(I. Saito & G. Stark, Proc. Natl. Acad. Sci. U.S.A., vol. 83, p8664-8668, 1986) のＫｐｎＩ、ＳｍａＩ、ＢａｍＨＩを除くため、charomid９−１１をＡｓｐ７１８とＢａｍＨＩで消化し、Ｋｌｅｎｏｗ酵素で平滑化後、セルフライゲーションした。これを用いて形質転換し、目的のシャロミドを単離し、charomid６−１１と名づけた。charomid６−１１のＥｃｏＲＩ部位へＢａｍＨＩリンカーを挿入し、得られたシャロミドをｃｈｄＲＢＲ７−１１と名づけた。
【００７８】
（vi）ｐＡｄｅｘ１ｃの調製
ｐＵＡＦ０−１７ＤのＢａｍＨＩ−Ｂｓｔ１１０７フラグメント（２．９ｋｂ）とアデノウイルスゲノムのＢｓｔ１１０７−ＥｃｏＲＩフラグメント（２１．６ｋｂ）とｐＸ２ＷのＥｃｏＲＩ−ＳｗａＩフラグメント（６．５ｋｂ）をＥｃｏＲＩとＥｃｌ３６Ｉで消化したｃｈｄＲＢＲ７−１１とライゲーションした。その後、in vitroパッケージングし、大腸菌ＤＨ５αへ感染させた。形質転換株から目的のフラグメントをもつものを単離し、ｐＡｄｅｘ１ｃと名づけた。
【００７９】
▲３▼ カセットコスミドｐＡｄｅｘ１ｃｗの構築
ＣｌａＩ（２０ｕｎｉｔ）で切断した後エタノール沈澱により回収したｐＡｄｅｘ１ｃ（１μｇ）と、５’末端リン酸化を施した下記の合成リンカー（１）（配列番号：２）０．０１μｇ（ＳｗａＩ、ＣｌａＩ、ＳａｌＩ、ＮｒｕＩ部位を含む）を混合し、ＡＴＰ、Ｔ４ＤＮＡリガーゼを含む反応液（最終容量１８μｌ）中で一晩結合させた。７０℃で１０分間インキュベートすることによりリガーゼを熱失活させた後、ＳｗａＩ（２０ｕｎｉｔ）を添加し、反応させた。この切断はリンカーが複数個挿入されたものからＳｗａＩ断片を切り出し、リンカーが１個のみ挿入された構造のコスミドを得るために行った。次に、反応液をＳｐｕｎ ｃｏｌｕｍｎ（Ｐｈａｒｍａｃｉａ製）にかけ、リンカー由来の小断片を除去した後、ＡＴＰ、Ｔ４ＤＮＡリガーゼを含む反応液（最終容量１８μｌ）中で一晩結合させ、セルフアニーリングにより環状化を行った。７０℃で１０分間インキュベートすることによりリガーゼを熱失活させ、１μｌをイン・ビトロ・パッケージングに用いた。次に、各コロニーから調製したコスミドＤＮＡの構造をＢａｍＨＩおよびＮｒｕＩ同時消化により確認した。目的とする方向に挿入された場合４８３ｂｐ、逆方向に挿入された場合、４６４ｂｐの断片を生じる。この確認により目的とするカセットコスミドｐＡｄｅｘ１ｃｗ（図１）を取得した。
合成リンカー

【００８０】
▲４▼ カセットコスミドｐＡｄｅｘｌｐＣＡｗの構築
▲１▼で構築したプラスミドｐＣＭｗＣＨ３１をＨｉｎｄIII およびＣｌａＩで同時消化し、Ｋｌｅｎｏｗ酵素により平滑化し、5'末端リン酸化を施したＰｍｅＩリンカーとのリガーゼ反応を行った。７０℃で１０分間インキュベートすることによりリガーゼを熱失活させた後、Ｐｓｐ１４０６Ｉを添加し、反応させた。この切断はリンカーが複数個挿入されたものからＰｓｐ１４０６Ｉ断片を切り出し、リンカーがＤＮＡ断片の両端にそれぞれ１個連結した構造の断片を得るために行った。このあと、反応液をアガロースゲル電気泳動に供し、２．３ｋｂのＤＮＡ断片を含むゲルを切り出し、電気泳動によりゲルからＤＮＡ断片を回収した。次に、ｐＡｄｅｘｌｃｗをＣｌａＩで切断した後、生じた小断片をＳｐｕｎ ｃｏｌｕｍｎ（Ｐｈａｒｍａｃｉａ製）により除去した後のＤＮＡ断片１μｇと前述の２．３ｋｂのＤＮＡ断片０．１μｇをＡＴＰ、Ｔ４ＤＮＡリガーゼを含む反応液（最終容量１８μｌ）中で一晩結合させた。７０℃で１０分間インキュベートすることによりリガーゼを熱失活させた後、これの１／４量にＣｌａＩを添加し（最終容量２０μｌ）、セルフアニーリングにより生じた環状コスミドを切断した。１μｌをイン・ビトロ・パッケージングに用いた。
次に、各コロニーから調製したコスミドＤＮＡの構造をＢａｍＨＩ及びＸｈｏＩ同時消化により確認した。目的とする方向に挿入された場合４８３ｂｐと４．８ｋｂ、逆方向に挿入された場合、２．７および２．５ｋｂの断片を生じる。この確認により目的とするカセットコスミドｐＡｄｅｘｌｐＣＡｗを取得した。
【００８１】
▲５▼ カセットコスミドｐＡｄｅｘｌＣＡｗｔ（細胞工学、Vol.１３、No.8、Ｐ７５９）の構築
ＳｗａＩ（２０ｕｎｉｔ）で切断した後エタノール沈澱により回収したｐＡｄｅｘｌｐＣＡｗ（１μｇ）と、5'末端リン酸化を施した下記の合成リンカー（２）（配列番号：３）（０．０１μｇ）（ＣｌａＩ、ＸｂａＩ、ＳｐｅＩ、ＰａｃＩ、ＳｗａＩ、ＣｌａＩ部位を含む）を混合し、ＡＴＰ、Ｔ４ＤＮＡリガーゼを含む反応液（最終容量１８μｌ）中で一晩結合させた。７０℃で１０分間インキュベートすることによりリガーゼを熱失活させた後、ＰａｃＩ（２０ｕｎｉｔ）を添加し、反応させた。この切断はリンカーが複数個挿入されたものからＰａｃＩ断片を切り出し、リンカーが１個のみ挿入された構造のコスミドを得るために行った。次に、反応液をＳｐｕｎ ｃｏｌｕｍｎ（Ｐｈａｒｍａｃｉａ製）にかけ、リンカー由来の小断片を除去した後、ＡＴＰ、Ｔ４ＤＮＡリガーゼを含む反応液（最終容量１８μｌ）中で一晩結合させ、セルフアニーリングによる環状化を行った。７０℃で１０分間インキュベートすることによりリガーゼを熱失活させた１μｌをイン・ビトロ・パッケージングに用いた。次に、各コロニーから調製したコスミドＤＮＡの構造をＸｂａＩおよびＸｈｏＩ同時消化により確認した。目的とする方向に挿入された場合５５２ｂｐ、逆方向に挿入された場合、５６８ｂｐの断片を生じる。これを確認することにより目的とするカセットコスミドｐＡｄｅｘｌＣＡｗｔ（図２）を取得した。
合成リンカーの構造

【００８２】
実施例２（ｌｏｘＰ挿入コスミドの構築）
▲１▼ ｐＡ６０Ｘ９９Ｘの作製
ｐＡｄｅｘｌＣＡｗｔ（０．５μｇ）をＢａｍＨＩ（１５ｕｎｉｔ）を含む反応系２０μｌ中で切断した後、熱処理（７０℃、１５分間）によりＢａｍＨＩを失活させた。次にその１／４量を用いリガーゼ反応ｂｕｆｆｅｒ中でＡＴＰ、Ｔ４ＤＮＡリガーゼを加え、最終容量２０μｌで一晩結合させた。次いでこの反応混液１０μｌを用いて大腸菌ＤＨ５αを形質転換し、得られた形質転換体からプラスミドＤＮＡを調製し、目的とするプラスミドｐＡ６０Ｘ９９Ｘを得た。
【００８３】
▲２▼ ｐＡ６０Ｘ９９の作製（アデノウイルス以外のＸｂａＩ部位を除去する）ｐＡ６０Ｘ９９Ｘ（５μｇ）をＸｂａＩ（１０ｕｎｉｔ）を含む反応系５０μｌ中で５分間反応させ、反応混液をアガロースゲル電気泳動し、２ヶ所のＸｂａＩ部位のうち１ヶ所のみで切断された２３．８ｋｂのＤＮＡ断片を含むゲルを切り出し、電気泳動によりゲルからＤＮＡ断片を回収した。次に、この断片０．２μｇをＫｌｅｎｏｗ酵素（宝酒造製）５ｕｎｉｔを含む反応系５０μｌ中で反応させ両末端を平滑化し、さらに、これの１／５量、ＡＴＰおよびＴ４ＤＮＡリガーゼを含む反応液（最終容量２０μｌ）中で一晩結合させた。次いでこの反応混液１０μｌを用いて大腸菌ＤＨ５αを形質転換し、得られた形質転換体からプラスミドＤＮＡを調製した。これらのプラスミドＤＮＡをＢｓｒＧＩおよびＸｂａＩで同時消化し、６．２ｋｂの断片を生じる、すなわち図１の構造を有するプラスミドｐＡ６０Ｘ９９（図３）を得た。
【００８４】
▲３▼ ｐＡ２Ｌ６０Ｘ９９の作製（ＢｓｒＧＩ部位へのｌｏｘＰの挿入）
ｐＵＬＬ２ｒ（３０μｇ）をＸｈｏｌ（１５０ｕｎｉｔ）を含む反応系１２５μｌ中で切断した後、熱処理（７０℃、１５分間）によりＸｈｏＩを失活させた。続いてＫｌｅｎｏｗ酵素（宝酒造製）１２ｕｎｉｔを含む反応系中で両末端を平滑化し、その後フェノール：クロロホルム（１：１）処理を施した後、エタノール沈澱した。沈澱物を遠心分離により取得し、１０ｍＭトリス−塩酸（ｐＨ７．５）に１ｍＭのＥＤＴＡを添加した溶液（ＴＥ）６０μｌに溶解した。次に、これの１／２量と５’末端リン酸化ＫｐｎＩリンカー（宝酒造製）０．２μｇ、ＡＴＰおよびＴ４ＤＮＡリガーゼを含むリガーゼ反応液（最終容量５０μｌ）中で一晩結合させた。次に、熱処理（７０℃、１５分間）によりリガーゼを失活させた後、Ａｓｐ７１８（１００ｕｎｉｔ）を含む反応系８０μｌ中で消化した。反応混液をアガロースゲル電気泳動し、ｌｏｘＰを含む６４ｂｐのＤＮＡ断片を含むゲルを切り出し、電気泳動によりゲルからＤＮＡ断片を回収した。
【００８５】
なお、上記のｐＵＬＬ２ｒは以下のようにして調製した。ｐＵＣ１１９（宝酒造製）を制限酵素Ｅｃｌ１３６IIで切断し、アルカリホスファターゼ処理を施した後、末端にＭｌｕＩ部位およびＸｈｏＩ部位を有しこれが連結するとＮｒｕＩ部位を生じるように設計されているｌｏｘＰ配列を含む下記の合成ＤＮＡ断片（配列番号：４）とのligation反応を行い該合成ＤＮＡ断片が２つ挿入されたプラスミドｐＵＬＬ２ｒを得た。

（下線部分の配列がｌｏｘＰ部位である。）
【００８６】
一方、プラスミドｐＡ６０Ｘ９９（１０μｇ）をＢｓｒＧＩ（５０ｕｎｉｔ）を含む反応系５０μｌ中で切断した後、反応混液をアガロースゲル電気泳動し、２３．８ｋｂのＤＮＡ断片を含むゲルを切り出し、電気泳動によりゲルからＤＮＡ断片を回収した。このＤＮＡ断片０．５μｇと前述のｌｏｘＰを含む６４ｂｐのＤＮＡ断片０．００５μｇ、ＡＴＰおよびＴ４ＤＮＡリガーゼを含むリガーゼ反応液（最終容量２５μｌ）中で一晩結合させた。これの１／２量に滅菌水、ＢｓｒＧＩ反応ｂｕｆｆｅｒを加えて１８μｌとしてから、７０℃で１０分間インキュベートすることによりリガーゼを熱失活させた。さらに、２０ｕｎｉｔのＢｓｒＧＩを加え（最終容量２０μｌ）３７℃で１時間反応させることによりセルフアニーリングにより生じた環状のｐＡ６０Ｘ９９を切断した。次いでこの反応混液１０μｌを用いて大腸菌ＤＨ５αを形質転換し、得られた形質転換体からプラスミドＤＮＡを調製した。
【００８７】
挿入されたｌｏｘＰの方向を確認するため、ＡｐａＩとＭｌｕＩの同時消化を行い、反応混液をアガロースゲル電気泳動した。目的とする方向に挿入された場合、３６６および２１９ｂｐ、逆方向に挿入された場合、３３４および２５１ｂｐの断片が生じる。また、ＮｒｕＩで消化した場合、目的とする方向に挿入された場合５７３ｂｐ、逆方向に挿入された場合、５３３ｂｐの断片が生じる。さらに、ＤｒａＩとＫｐｎＩで同時消化した場合、目的とする方向にｌｏｘＰが１つ挿入された場合３２０ｂｐ、２つ挿入された場合、３８４ｂｐの断片が生じる。これら３種の条件をすべて満たす、すなわち、目的とする方向にｌｏｘＰが１つ挿入された目的のプラスミドｐＡ２Ｌ６０Ｘ９９（図４）を取得した。
【００８８】
▲４▼ ｐＡ２Ｌ３Ｌ６０９９の作製（ＸｂａＩ部位へのｌｏｘＰの挿入）
ｐＵＬＬ２ｒ（２０μｇ）をＸｈｏＩ（１００ｕｎｉｔ）を含む反応系１００μｌ中で切断した後、熱処理（７０℃、１５分間）によりＸｈｏＩを失活させた。続いてＫｌｅｎｏｗ酵素（宝酒造製）８ｕｎｉｔを含む反応系において両末端を平滑化し、その後フェノール：クロロホルム（１：１）処理を施した後、エタノール沈澱した。沈澱物を遠心分離により取得し、ＴＥ３０μｌに溶解した。これの全量と５’末端リン酸化ＳｐｅＩリンカー（宝酒造製）０．４μｇ、ＡＴＰおよびＴ４ＤＮＡリガーゼを含む反応液（最終容量５０μｌ）中で一晩結合させ、７０℃で１０分間インキュベートすることによりリガーゼを熱失活させた。さらに、ＳｐｅＩ（５４ｕｎｉｔ）を加え消化した後、反応混液をアガロースゲル電気泳動し、ｌｏｘＰを含む６４ｂｐのＤＮＡ断片を含むゲルを切り出し、電気泳動によりゲルからＤＮＡ断片を回収した。
【００８９】
一方、ｐＡ２Ｌ６０Ｘ９９（１０μｇ）を、ＸｂａＩ（１００ｕｎｉｔ）を含む反応系５０μｌにおいて切断した後、反応混液をアガロースゲル電気泳動し、２３．８ｋｂのＤＮＡ断片を含むゲルを切り出し、電気泳動によりゲルからＤＮＡを回収した。このＤＮＡ断片０．５μｇと前述のｌｏｘＰを含む６４ｂｐのＤＮＡ断片０．００５μｇ、ＡＴＰおよびＴ４ＤＮＡリガーゼを含む反応液（最終容量１６μｌ）中で一晩結合させた。これに５倍希釈したＴＥ１４μｌを加え、７０℃で１０分間インキュベートすることによりリガーゼを熱失活させた。次いでこれの１／４量に２０ｕｎｉｔのＸｂａＩを添加し（最終容量２０μｌ）、セルフアニーリングにより生じた環状のｐＡ２Ｌ６０Ｘ９９を切断した。この反応混液１０μｌを用いて大腸菌ＤＨ５αを形質転換し、得られた形質転換体からプラスミドＤＮＡを調製した。
【００９０】
挿入されたｌｏｘＰの方向を確認するため、ＢｇｌIIとＭｌｕＩの同時消化を行い、反応混液をアガロースゲル電気泳動した。目的とする方向に挿入された場合、３６６および５０３ｂｐ、逆方向に挿入された場合、３９８および４７１ｂｐの断片が生じる。また、ＡｐａＩとＭｌｕＩで同時消化した場合、目的とする方向に挿入された場合６６０ｂｐ、逆方向に挿入された場合、６２８ｂｐの断片が生じる。ＥｃｏＮＩとＭｌｕＩで消化した場合、目的とする方向に挿入された場合３１１ｂｐ、逆方向に挿入された場合、３４３ｂｐの断片が生じる。さらに、ＨｐａＩとＳａｃＩで同時消化した場合、目的とする方向にｌｏｘＰが１つ挿入された場合３９７ｂｐ、２つ挿入された場合、４６１ｂｐの断片が生じる。これら４種の条件をすべて満たす、すなわち、目的とする方向にｌｏｘＰが１つ挿入された目的のプラスミドｐＡ２Ｌ３Ｌ６０９９（図５）を取得した。
【００９１】
▲５▼ ｐＡｄｅｘ２Ｌ３ＬＣＡｗｔの作製
ｐＡｄｅｘ１ＣＡｗｔ（１０μｇ）を、Ｃｓｐ４５Ｉ（４０ｕｎｉｔ）を含む反応系１００μｌ中で切断し、次いで、同反応液中にＢａｍＨＩ（３０ｕｎｉｔ）、さらにＥｃｏＲＩ（４０ｕｎｉｔ）を順次添加した。２１ｋｂのＤＮＡ断片を含むゲルを切り出し、電気泳動によりゲルからＤＮＡ断片を回収した。なお、Ｃｓｐ４５１およびＥｃｏＲＩによる切断は２１ｋｂのＢａｍＨＩＤＮＡ断片を回収する際、他の断片が混入するのを防ぐためである。
【００９２】
ｐＡ２Ｌ３Ｌ６０９９（５μｇ）を、ＢａｍＨＩ（３０ｕｎｉｔ）を含む反応系５０μｌ中で切断した後フェノール：クロロホルム（１：１）処理を施し、水層をＴＥで平衡化したＳｅｐｈａｄｅｘＧ２５によりゲル濾過した。回収したＤＮＡ断片０．５μｇと前述の２１ｋｂのＤＮＡ断片０．５μｇ、ＡＴＰおよびＴ４ＤＮＡリガーゼを含む反応液（最終容量１８μｌ）中で一晩結合させた。７０℃で１０分間インキュベートすることによりリガーゼを熱失活させた後、１μｌをイン・ビトロ・パッケージングに用いた。
【００９３】
即ち、ラムダ・イン・ビトロ・パッケージングキットであるギガバックＸＬ（Ｓｔｒａｔａｇｅｎｅ社製）を１／４スケールで用い、残りは−８０℃に凍結した。ギガバックＸＬは４２ｋｂ以下のコスミドのパッケージ効率が低いのでインサートが入って大きくなったコスミドをある程度選択することができる。本実験では、１０個のコロニーを拾えば大半はインサートを含んでおり、目的のクローン（ウイルスゲノムが正しく連結されたクローン）を容易に得ることができた。コスミドの扱い方については、常法（斎藤 泉他、実験医学：７：183-187, 1989)に従って行った。
【００９４】
パッケージングされたコスミドを大腸菌ＤＨ５α（ＧｉｂｃｏＢＲＬ）に感染させた。即ち、３枚のＡｐ⁺ （アンピシリン添加）寒天プレートと５ｍｌのＡｐ⁺ ＬＢ（ｐｏｏｌ）にそれぞれ１／２００量、１／２０量、１／２量、残り全量を接種し、一晩培養した。
ｐｏｏｌのｍｉｎｉｐｒｅｐＤＮＡを抽出・調製し、制限酵素ＤｒａＩ切断によりインサートがはいったものの割合を調べた。コロニーは丸ごと寒天ごと取り１．５ｍｌのＡｐ⁺ ＬＢで、一晩培養し、ｍｉｎｉｐｒｅｐＤＮＡを調製した。次に、各コロニーから調製したコスミドＤＮＡの構造をＤｒａＩ切断により確認した。目的とする方向に挿入された場合８９１ｂｐ、逆方向に挿入された場合１．４ｋｂの断片を生じる。これにより目的とするカセットコスミドｐＡｄｅｘ２Ｌ３ＬＣＡｗｔを取得した。
【００９５】
実施例３
（アデノウイルスＤＮＡ−末端蛋白複合体（Ａｄ５ ｄｌＸ ＤＮＡ−ＴＰＣおよびＡｄｅｘ１ＣＡＮＬａｃＺ ＤＮＡ−ＴＰＣ）の調製）
▲１▼ アデノウイルスＤＮＡとしては、Ａｄ５ ｄｌＸ（I. Saito et al., J.Virology, vol.54, 711-719 (1985))またはＡｄｅｘ１ＣＡＮＬａｃＺを用いる。Ａｄ５ ｄｌＸをＨｅＬａ細胞（Ｒｏｕｘ １０本分）にまたＡｄｅｘ１ＣＡＮＬａｃＺを２９３細胞にそれぞれ感染させ、培養を行った。
即ち、Ａｄ５−ｄｌＸまたはＡｄｅｘ１ＣＡＮＬａｃＺのウイルス液（〜１０⁹ ＰＦＵ／ｍｌ）を０．２ｍｌ／Ｒｏｕｘ感染させ、３日後に、はがれた細胞を遠心分離により集めた。アデノウイルス粒子のほとんどはメディウム中ではなく細胞の核内にいるので感染細胞からウイルスを精製できる利点がある。（以下の操作は非無菌的に行う。）
【００９６】
▲２▼ 得られた細胞をＴｒｉｓ−ＨＣｌ（ｐＨ８．０）に懸濁し、密封型ソニケーターを用いて細胞を破砕し、ウイルスを細胞内から放出させた。
▲３▼ 得られた破砕物を遠心分離により沈澱を除いた後、超遠心分離機 ＳＷ２８チューブに塩化セシウム溶液（比重１．４３）を入れ、その上に上清を重層し、クッション遠心による濃縮を行った。
【００９７】
▲４▼ 界面直下のウイルス層をＳＷ５０．１チューブに移した。界面直下のウイルス層は通常目視でき、ウイルス層とその下層の塩化セシウムを５ｍｌ採取した。同時にもう一本に塩化セシウム溶液（比重１．３４）を満たした。
これらを、３５ｋｒｐｍ、４℃で一晩超遠心にかけた。次いで、白いウイルスのバンドを分取し、既に勾配ができたチューブに乗せ替えた。さらに、３５ｋｒｐｍ、４℃４時間以上超遠心にかけた。
【００９８】
▲５▼ 白いウイルスのバンドを分取し、等量の８Ｍ塩酸グアニジンと室温で混合し、４Ｍ塩酸グアニジン飽和塩化セシウムを加えてＶＴｉ６５チューブに満たした。４Ｍ塩酸グアニジンにより、粒子蛋白は変性を受けて解離し、ＤＮＡ−ＴＰＣが放出された。
【００９９】
▲６▼ 上記のチューブを、５５ｋｒｐｍ、１５℃で一晩超遠心にかけ、０．２ｍｌずつ分画し、その１μｌずつを１μｇ／ｍｌのエチジウムブロミド水溶液２０μｌと混合し、蛍光染色することによりＤＮＡの有無を確認する。ＤＮＡを含む２〜３フラクションを集めた。
▲７▼ ５００ｍｌのＴＥに一晩透析（２回）し、−８０℃に保存した。こうして得られたＡｄ５ｄｌＸおよびＡｄｅｘ１ＣＡＮＬａｃＺ ＤＮＡ−ＴＰＣの量をＯＤ₂₆₀ から通常のＤＮＡと同様に算出した。
▲８▼ 得られたＡｄ５ｄｌＸおよびＡｄｅｘ１ＣＡＮＬａｃＺ ＤＮＡ−ＴＰＣを、第３ステップの組換えアデノウイルス作成のため、充分量のＡｇｅＩで２時間切断し、Ｓｅｐｈａｄｅｘ Ｇ２５カラムでゲル濾過後、−８０℃に保存した。
【０１００】
なお、ＤＮＡ−ＴＰＣは制限酵素による切断、透析、ゲル濾過はできるが電気泳動・フェノール処理・エタノール沈澱はできない。濃縮法は塩化セシウム平衡遠心しかないのでなるべく濃厚状態に保った。１０Ｒｏｕｘの感染細胞から約３００μｇ程度のＤＮＡ−ＴＰＣを得ることができた。
▲９▼ 一部を分取し、泳動用ＢＰＢ ｂｕｆｆｅｒを１０μｌ加えた後に、１μｌのプロテイナーゼＫ（１０ｍｇ／ｍｌ）を加えて３７℃で１０分間反応させて末端蛋白を消化した。フェノール抽出し、上清をアガロースゲル電気泳動で分離し、完全切断を確認した。
ＡｇｅＩ切断ＤＮＡ−ＴＰＣ中の制限酵素ｂｕｆｆｅｒを、遠心ゲル濾過によって除いた後、分注し−８０℃に保存した。
【０１０１】
実施例４
（ｌｏｘＰ挿入組み換えアデノウイルス（Ａｄ５ｄｌＸ２Ｌ３ＬまたはＡｄｅｘ２Ｌ３ＬＣＡＮＬａｃＺ）の作製）
なお、ＮＬａｃＺは大腸菌ＬａｃＺ遺伝子のＮ末端にＳＶ４０の核移行シグナルを付加したものである。
▲１▼ １０％ＦＣＳ添加ＤＭＥで培養した２９３細胞の６ｃｍ、１０ｃｍシャーレ各１枚用意した。
▲２▼ 発現ユニットを組み込んだｌｏｘＰを挿入したコスミドｐＡｄｅｘ２Ｌ３ＬＣＡｗｔ ＤＮＡの８μｇとＡｇｅＩで切断したＡｄ５ｄｌＸ ＤＮＡ−ＴＰＣまたはＡｇｅＩで切断したＡｄｅｘ１ＣＡＮＬａｃＺ ＤＮＡ−ＴＰＣの１μｇを混合し、セルフェクト（ファルマシア製）キットを用いて、６ｃｍシャーレ１枚にリン酸カルシウム法でトランスフェクションを行った。６ｃｍシャーレのメディウムの上から混合液を滴下し、培養を続けた。
一晩培養（約１６時間）し、午前中に培養液を交換し、夕方、コラーゲンコート９６穴３枚（原液・１０倍希釈・１００倍希釈）に、５％ＦＣＳ添加ＤＭＥを用い、各ウエル当たり０．１ｍｌでまき直した。細胞数が各プレートで大きく違わないように、希釈２枚分には１０ｃｍシャーレの２９３細胞を１／３ずつ混ぜて播いた。
【０１０２】
▲３▼ ３〜４日後と８〜１０日後に、各ウエルに５０μｌの１０％ＦＣＳ添加ＤＭＥを加えた。２９３細胞がやせてきたら早めに加えた。
ウイルスが増殖し細胞が死滅したウエルが７〜２０日の間に現れた。ウエルの細胞が完全に死滅するごとに滅菌パスツールピペットで培養液（死細胞ごと）を滅菌した１．５ｍｌチューブに無菌的に移して、ドライアイスで急凍して−８０℃に保存した。
▲４▼ １５〜２５日で判定は終了した。比較的遅く細胞が死んだウエルから回収した培養液チューブを約１０個選び、超音波破砕後、５０００ｒｐｍ１０分遠心分離して得られた上清を１次ウイルス液（first seed）として−８０℃に保存した。
早めにウイルス増殖が起こったウエルは複数のウイルス株の混合感染の可能性が高いからである。
【０１０３】
▲５▼ ２４穴プレートに２９３細胞を用意し、５％ＦＣＳ−ＤＭＥ（０．４ｍｌ／ウエル）と１次ウイルス液１０μｌをそれぞれ２ウエルずつ添加した。
▲６▼ 約３日で細胞が完全に死滅したら、１ウエルは１次ウイルス液作製と同様に超音波破砕と遠心分離で上清を得、これを２次ウイルス液(second seed) として−８０℃に保存した。他の１ウエルの死滅した細胞を５０００ｒｐｍで５分間遠心し、上清を捨てて細胞だけを−８０℃に保存した（セルパック）。１０種類のウイルス株のセルパックが集まったら以下の方法で感染細胞の全ＤＮＡを抽出した。セルパックには、４００μｌのcell DNA用TNE (50mM Tris-HCl pH7.5, 100mM NaCl, 10mM EDTA)、４μｌのproteinaseK (10mg/ml) および４μｌの10%SDSを加えた。
【０１０４】
▲７▼ ５０℃で１時間処理した後、フェノール・クロロホルム抽出２回、クロロホルム抽出２回、ついでエタノール沈澱により得られた核酸をＲＮａｓｅを２０μｇ／ｍｌ含む５０μｌのＴＥに溶かした。
その１５μｌを発現ユニットを切断する酵素の中で認識配列にＣＧを含む酵素であるＸｈｏＩで切断し、発現コスミドカセットのＸｈｏＩ切断と共に、１５ｃｍ位の長さのアガロースゲルで一晩電気泳動を行い、パターンを比較した。ＸｈｏＩは挿入したｌｏｘＰ配列内に認識部位があるので、ｌｏｘＰが２個挿入された切断パターンを示すクローンを選択する。説明できないバンドが薄く見えるクローンは、欠失のあるウイルスとの混合の可能性があるので廃棄した。
【０１０５】
実施例５
（Ｅ２Ａ遺伝子欠損アデノウイルスの作製と構造確認）
組み換えアデノウイルスＡｄｅｘ２Ｌ３ＬＣＡＮＬａｃＺおよびＡｄｅｘ１ＣＡＮＣｒｅをそれぞれｍｏｉ＝１０および３で２９３細胞に感染させ、培養を行った。なお、ＮＣｒｅは、ＮＬａｃＺと同様、ＳＶ４０の核移行シグナルをＣｒｅのＮ末端に付加したものである。
４日目に細胞を回収し、前述の方法によりＤＮＡを調製した。ｌｏｘＰで挟まれた領域（Ｅ２Ａ遺伝子を含む）が切り出された構造を有するＡｄｅｘｄ１２３ＣＡＮＬａｃＺの生成を２つの方法で確認した。
【０１０６】
１．ＳｍａＩ消化
ＳｍａＩ消化の後、ゲル電気泳動した結果、ｌｏｘＰで挟まれた領域が切り出されて生じる４．７ｋｂの断片が認められた。このバンドとＡｄｅｘ２Ｌ３ＬＣＡＮＬａｃＺ、Ａｄｅｘ１ＣＡＮＣｒｅ、およびＡｄｅｘｄ１２３ＣＡＮＬａｃＺにおいて共通して見られる４．４５ｋｂのバンドの濃さの比較から、回収した組換えアデノウイルス中の約３０％がＡｄｅｘｄ１２３ＣＡＮＬａｃＺであることが分かった。
【０１０７】
２．ＰＣＲによる確認
調製したＤＮＡ０．１ｎｇをテンプレートとし、下記の条件でＰＣＲを行い、生成物をアガロースゲル電気泳動により分析した。用いたプライマーは、下記に示すオリゴヌクレオチド（１）（配列番号：５）、オリゴヌクレオチド（２）（配列番号：６）、オリゴヌクレオチド（３）（配列番号：７）およびオリゴヌクレオチド（４）（配列番号：８）である。

ＰＣＲ反応液組成（総容量２０μｌ）
Ｔｒｉｓ・ＨＣｌ（ｐＨ８．３） １０ ｍＭ
ＫＣｌ ５０ ｍＭ
ＭｇＣｌ₂ １．５ｍＭ
ｄＮＴＰ mixture ０．２ｍＭ
プライマー 各０．２μＭ
テンプレートＤＮＡ ０．１ｎｇ
Ｔａｑ polymerase ０．５ｕｎｉｔ
ＰＣＲ反応条件
二本鎖解離 ： ９５℃ １．５分
アニーリング： ６４℃ １．０分
伸長反応 ： ７０℃ １．０分
反応サイクル： ３０回
その結果を図６に示す。
プライマーとして（１）と（４）を用いた場合、３９３ｂｐと推定されるバンドが検出され、Ｅ２Ａ遺伝子が欠失したＡｄｅｘｄ１２３ＣＡＮＬａｃＺの存在が明らかとなった（図６、レーン１）。
また、（２）と（３）を用いた場合、２２１ｂｐと推定されるバンドが検出され、Ｃｒｅ遺伝子産物により切り出された環状のＥ２Ａ遺伝子の存在が裏付けられた（図６、レーン２）。
以上、１および２の結果から、Ａｄｅｘ２Ｌ３ＬＣＡＮＬａｃＺからｌｏｘＰではさまれた領域（Ｅ２Ａ遺伝子を含む）が除去されたＡｄｅｘｄ１２３ＣＡＮＬａｃＺの生成が明らかになった。
【０１０８】
参考例１
＜リコンビナーゼＣｒｅ遺伝子およびＣＡＧプロモーターを有する組換えアデノウイルスベクターの作製＞
（１）リコンビナーゼＣｒｅ遺伝子発現用カセットコスミドの作製
▲１▼ リコンビナーゼＣｒｅ遺伝子を含む大腸菌ファージＰ１ＤＮＡ（ＡＴＣＣ１１３０３−Ｂ２３）をテンプレートとし、５’−プライマーとして下記の（配列番号：９）のオリゴヌクレオチドを、３’−プライマーとして下記の（配列番号：１０）のオリゴヌクレオチドを、耐熱性ポリメラーゼとしてＮＥＢ社製のＶｅｎｔ^R を用い、以下の条件でＰＣＲ反応を行い、生成物をアガロースゲル電気泳動にかけ、約１ｋｂのバンドを切り出し、リコンビナーゼＣｒｅ遺伝子を含む約１ｋｂのＤＮＡ断片を得た。

（下線部分は、制限酵素の認識部位である。）
【０１０９】
ＰＣＲ反応条件
緩衝液： １０ｍＭのＫＣｌ、２０ｍＭのＴｒｉｓ−ＨＣｌ（ｐＨ８．８）、１０ｍＭの（ＮＨ₄ ）₂ ＳＯ₄ 、２ｍＭのＭｇＳＯ₄ 、０．１％のＴｒｉｔｏｎ Ｘ−１００（ＮＥＢ社添付の緩衝液を使用）
耐熱性ポリメラーゼ： ２ユニット
ｄＮＴＰ： ４００μＭ
プライマー： １μＭ
Ｐ１ファージＤＮＡ： １ｎｇ
２本鎖解離温度： ９５℃ １．５分間
アニーリング温度： ６０℃ １．５分間
伸長反応温度： ７４℃ ２．０分間
反応サイクル： ２０回
【０１１０】
この断片およびｐＵＣ１９（宝酒造製）をそれぞれ制限酵素ＰｓｔＩ（宝酒造製）およびＸｂａＩ（宝酒造製）により同時消化したのち、回収し、モル比が約３：１になるように混合し、Ｔ４ＤＮＡリガーゼ（宝酒造製）を用いてligation反応を行った。さらに、この反応混液を用いて大腸菌ＪＭ１０９株（ＡＴＣＣ５３３２３）を形質転換した。アンピシリン（１００μｇ／ｍｌ）を添加したＬＢ寒天プレートから形質転換株を拾い、リコンビナーゼＣｒｅ遺伝子を含むプラスミドｐＵＣＣｒｅを得た。
【０１１１】
次に、ＣＡＧプロモーターを含むカセットコスミドｐＡｄｅｘ１ＣＡｗｔをＳｗａＩで切断したもの１μｇと、ｐＵＣＣｒｅをＰｓｔＩおよびＸｂａＩにより同時消化し、さらにＫｌｅｎｏｗ酵素（宝酒造製）により両端を平滑化して得た約１ｋｂの断片０．１μｇとを混合した。
【０１１２】
▲２▼ 次に、混合液にエタノールを加えてコスミドを沈澱させた。沈澱物を遠心分離により取得し、１０ｍＭトリス−塩酸（ｐＨ７．５）に１ｍＭのＥＤＴＡを添加した溶液（ＴＥ）の５倍希釈液に溶解した。
▲３▼ 得られたコスミドをリガーゼ反応ｂｕｆｆｅｒ中でＡＴＰ，Ｔ４ＤＮＡリガーゼを加え、最終容量７μｌで一晩結合させた。ついで滅菌水、ＳｗａＩ反応ｂｕｆｆｅｒを加えて４８μｌとしてから７０℃１０分でリガーゼを熱失活させた。
この際、プラズミドと異なり、コスミドでは、環状ではなく直鎖状タンデムに結合した巨大分子が効率よくパッケージされる。
【０１１３】
▲４▼ ２μｌのＳｗａＩ（Ｂｏｅｈｒｉｎｇｅｒ製）を加え、２５℃で１時間切断した。
ＳｗａＩ切断を行う意味は、カセットコスミドが発現ユニットをくわえ込むことなく再結合するとＳｗａｌ認識配列が再生されるため、このステップで発現ユニットの組み込まれていないコスミドを再切断し、コロニーを作らなくするためである。この方法はインサートをもつカセットコスミドだけを選択する強力な方法である。
▲５▼ 常法（Molecular Cloning vol.3 E.34）に従い、カセットコスミドのフェノール抽出、遠心分離、ついでゲル濾過を行った。
▲６▼ 再度、Ｓｗａｌ切断を行った。即ち、ＳｗａＩ反応ｂｕｆｆｅｒ中、５μｌのＳｗａＩを加え、２５℃で２時間切断した。その理由は上記の通りである。
【０１１４】
▲７▼ 得られたコスミドの１μｌについてイン・ビトロ・パッケージングを行った。
即ち、ラムダ・イン・ビトロ・パッケージングキットであるギガバックＸＬ（Ｓｔｒａｔａｇｅｎｅ製）を１／４スケールで用い、残りは−８０℃に凍結した。ギガバックＸＬは４２ｋｂ以下のコスミドのパッケージ効率が低いのでインサートが入って大きくなったコスミドをある程度選択することができる。本実験では、１０個のコロニーを拾えば大半はインサートを含んでおり、目的の向き（左向き）のクローンを容易に得ることができた。
コスミドの扱い方については、常法（斎藤 泉他、実験医学：７：183-187, 1989)に従って行った。
【０１１５】
▲８▼ パッケージングされたコスミドを大腸菌ＤＨ１（ＡＴＣＣ３３８４９）に感染させた。
即ち、３枚のＡｐ⁺ （アンピシリン添加）寒天プレートと５ｍｌのＡｐ⁺ ＬＢ（ｐｏｏｌ）にそれぞれ１／２００量、１／２０量、１／２量、残り全量を接種し、一晩培養した。
ｐｏｏｌのｍｉｎｉｐｒｅｐＤＮＡを抽出・調製し、全酵素切断によりインサートが入ったものの割合を調べた。コロニーは丸ごと寒天ごと取り１．５ｍｌのＡｐ⁺ ＬＢで、一晩培養し、ｍｉｎｉｐｒｅｐＤＮＡを調製した。
▲９▼ 次に、制限酵素切断により、発現ユニットの向きと構造を確認した。
なお、ＮｒｕＩとリガーゼを用いて、発現単位を含むが大部分のアデノウイルスＤＮＡを欠失したプラスミドを作製し、ＤＮＡを調製して、ｃＤＮＡクローン化の最終確認をした。
【０１１６】
（２）アデノウイルスＤＮＡ−末端蛋白複合体（Ａｄ５ ｄｌＸ ＤＮＡ−ＴＰＣ）の調製
▲１▼ アデノウイルスＤＮＡとしては、Ａｄ５ ｄｌＸ（I. Saito et al., J.Virology, vol.54, 711-719 (1985))を用いた。Ａｄ５ ｄｌＸを２９３細胞（Ｒｏｕｘ １０本分）に感染させ、培養を行った。
即ち、Ａｄ５−ｄｌＸのウイルス液（〜１０⁹ ＰＦＵ／ｍｌ）を０．２ｍｌ／Ｒｏｕｘ感染させ、３日後に、はがれた細胞を１５００ｒｐｍ、５分にて遠心分離して集めた。アデノウイルス粒子のほとんどはメディウム中ではなく細胞の核内にいるので感染細胞からウイルスを精製できる利点がある。（以下の操作は非無菌的に行った。）
【０１１７】
▲２▼ 得られた細胞を１０ｍＭのＴｒｉｓ−ＨＣｌ（ｐＨ８．０）の２０ｍｌに懸濁し、密封型ソニケーターを用い、２００Ｗ、２分（３０秒×４）で細胞を破砕し、ウイルスを細胞内から放出させた。
ウイルスを細胞内から放出させるには５ｍｌ以下なら凍結融解５回でもよいが、それ以上の容量ではソニケーターが便利である。ただし、必ず密封型（専用カップのあるもの）を用いる。通常の投げ込み型は、たとえ安全キャビネットの中でも危険性がある。
【０１１８】
▲３▼ 得られた破砕物を遠心分離（１０ｋｒｐｍ、１０分）により沈澱を除いた後、超遠心機 ＳＷ２８チューブに１５ｍｌの塩化セシウム溶液（比重１．４３）を入れ、その上に上清を重層し、クッション遠心（２５ｋｒｐｍ、１時間、４℃）による濃縮を行った。
▲４▼ 界面直下のウイルス層をＳＷ５０．１チューブに移した。界面直下のウイルス層は通常目視でき、ウイルス層とその下層の塩化セシウムを５ｍｌ採取した。同時にもう一本に塩化セシウム溶液（比重１．３４）を満たした。
これらを、３５ｋｒｐｍ、４℃で一晩超遠心にかけた。次いで、白いウイルスのバンドを分取し、既に勾配ができたチューブに乗せ替えた。さらに、３５ｋｒｐｍ、４℃４時間以上超遠心にかけた。
【０１１９】
▲５▼ 白いウイルスのバンドを分取し、等量の８Ｍ塩酸グアニジンと室温で混合し、４Ｍ塩酸グアニジン飽和塩化セシウムを加えてＶＴｉ６５チューブに満たした。４Ｍ塩酸グアニジンにより、粒子蛋白は変性を受けて解離し、ＤＮＡ−ＴＰＣが放出された。エチジウムブロミドは後で除く方法が確立されていないため利用できなかった。
【０１２０】
▲６▼ 上記のチューブを、５５ｋｒｐｍ、１５℃で一晩超遠心にかけ、０．２ｍｌずつ分画し、その１μｌずつを１μｇ／ｍｌのエチジウムブロミド水溶液２０μｌと混合し、蛍光染色することによりＤＮＡの有無を確認した。ＤＮＡを含む２〜３フラクションを集めた。
▲７▼ ５００ｍｌのＴＥに一晩透析（２回）し、−８０℃に保存した。こうして得られたＡｄ５ｄｌＸ ＤＮＡ−ＴＰＣの量をＯＤ₂₆₀ から通常のＤＮＡと同様に算出した。
【０１２１】
▲８▼ 得られたＡｄ５ｄ１Ｘ ＤＮＡ−ＴＰＣを、第３ステップの組換えアデノウイルス作成のため、充分量のＥｃｏＴ２２Ｉで２時間切断した後、−８０℃に保存した。
【０１２２】
なお、ＤＮＡ−ＴＰＣは制限酵素による切断、透析、ゲル濾過はできるが電気泳動・フェノール処理・エタノール沈澱はできなかった。濃縮法は塩化セシウム平衡遠心しかないのでなるべく濃厚状態に保った。１０Ｒｏｕｘの感染細胞から約３００μｇ程度のＤＮＡ−ＴＰＣを得ることができた。
▲９▼ 一部を分取し、泳動用ＢＰＢ ｂｕｆｆｅｒを１０μｌ加えた後に、１μｌのプロテイナーゼＫ（１０ｍｇ／ｍｌ）を加えて３７℃で１０分間反応させて末端蛋白を消化した。フェノール抽出し、上清をアガロースゲル電気泳動で分離し、完全切断を確認した。
ＥｃｏＴ２２Ｉ切断ＤＮＡ−ＴＰＣ中の制限酵素ｂｕｆｆｅｒを、遠心ゲル濾過によって除いた後、分注し−８０℃に保存した。
【０１２３】
（３）組換えウイルスの分離と高力価ウイルス液の作製
▲１▼ １０％ＦＣＳ添加ＤＭＥで培養した２９３細胞の６ｃｍ、１０ｃｍシャーレ各１枚用意した。
▲２▼ 発現ユニットを組み込んだｐＡｄｅｘ１ｗ ＤＮＡの８μｇ（３〜９μｇが適当である）とＥｃｏＴ２２Ｉで切断したＡｄ５ｄｌＸ ＤＮＡ−ＴＰＣの１μｇを混合し、セルフェクト（ファルマシア製）キットを用いて、６ｃｍシャーレ１枚にリン酸カルシウム法でトランスフェクションを行った。６ｃｍシャーレのメディウムの上から混合液を滴下し、培養を続けた。
一晩培養（約１６時間）し、午前中に培養液を交換し、夕方、コラーゲンコート９６穴３枚（原液・１０倍希釈・１００倍希釈）に、５％ＦＣＳ添加ＤＭＥを用い、各ウエル当たり０．１ｍｌでまき直した。細胞数が各プレートで大きく違わないように、希釈２枚分には１０ｃｍシャーレの２９３細胞を１／３ずつ混ぜて播いた。
【０１２４】
▲３▼ ３〜４日後と８〜１０日後に、各ウエルに５０μｌの５％ＦＣＳ添加ＤＭＥを加えた。２９３細胞がやせてきたら早めに加えた。
ウイルスが増殖し細胞が死滅したウエルが７〜１５日の間に現れた。ウエルの細胞が完全に死滅するごとに滅菌パスツールピペットで培養液（死細胞ごと）を滅菌した１．５ｍｌチューブに無菌的に移して、ドライアイスで急凍して−８０℃に保存した。
▲４▼ １５〜１８日で判定は終了した。比較的遅く細胞が死んだウエルから回収した培養液チューブを約１０個選び、凍結融解６回後、５ｋｒｐｍ１０分遠心して得られた上清を１次ウイルス液（first seed）として−８０℃に保存した。
早めにウイルス増殖が起こったウエルは複数のウイルス株の混合感染の可能性が高いからである。
【０１２５】
▲５▼ ２４穴プレートに２９３細胞を用意し、５％ＦＣＳ−ＤＭＥ（０．４ｍｌ／ウエル）と１次ウイルス液１０μｌをそれぞれ２ウエルずつ添加した。
▲６▼ 約３日で細胞が完全に死滅したら、１ウエルは１次ウイルス液作製と同様に６回の凍結融解と遠心で上清を得、これを２次ウイルス液(second seed) として−８０℃に保存した。２次ウイルス液の力価は１０⁷ 〜１０⁸ ＰＦＵ／ｍｌ程度であった。他の１ウエルの死滅した細胞を５ｋｒｐｍで５分間遠心し、上清を捨てて細胞だけを−８０℃に保存した（セルパック）。１０種類のウイルス株のセルパックが集まったら以下の方法で感染細胞の全ＤＮＡを抽出した。セルパックには、４００μｌのcell DNA用TNE (50mM Tris-HCl pH7.5, 100mM NaCl, 10mM EDTA)、４μｌのproteinaseK (10mg/ml) および４μｌの10%SDSを加えた。
【０１２６】
▲７▼ ５０℃で１時間処理した後、フェノール・クロロホルム抽出２回、クロロホルム抽出２回、ついでエタノール沈澱により得られた核酸をＲＮａｓｅを２０μｇ／ｍｌ含む５０μｌのＴＥに溶かした。
その１５μｌを発現ユニットを切断する酵素の中で認識配列にＣＧを含む酵素であるＸｈｏＩで切断し、発現コスミドカセットのＸｈｏＩ切断と共に、１５ｃｍ位の長さのアガロースゲルで一晩電気泳動を行い、パターンを比較した。発現ユニット内の切断点からアデノウイルスゲノムの左端までのバンドが正確に出現しているものを選択した。また、説明できないバンドが薄く見えるクローンは、欠失のあるウイルスとの混合の可能性があるので廃棄した。
アデノウイルスＤＮＡは細胞あたり１０，０００コピーに増殖するので、細胞ＤＮＡと一緒に全ＤＮＡを抽出し制限酵素切断によりウイルスＤＮＡのバンドをみることができる。Ｘｈｏｌなどのように認識配列にＣＧを含む酵素は、細胞ＤＮＡを切断しないので、パターンが見やすい。これ以外の酵素を用いるときは、非感染２９３細胞ＤＮＡをコントロールにおくことが必要であった。（ヒト細胞の反復配列由来のバンドが出現した）。
【０１２７】
▲８▼ Ｘｈｏｌ切断で同定された目的のウイルス株の２次ウイルス液の０．１ｍｌを、コラーゲンコートした１５０ｃｍ² ボトル（培地は２５ｍｌ）の２９３細胞へ感染させた。
３日後に細胞が死滅したら、死細胞ごと２５ｍｌの培地を無菌的に密閉型ソニケーター２００ｗ最高出力２分（３０秒×４回）で破砕してウイルスを遊離させた。
３ｋｒｐｍ、４℃で１０分間遠心して沈澱を除去し、５ｍｌ凍結用チューブに２ｍｌずつ１３本に分注し、ドライアイスで急凍して−８０℃に保存し、３次ウイルス液を調製した。３次ウイルス液は本発明の組換えアデノウイルスを含む液であり、１０⁹ ＰＦＵ／ｍｌ程度の高力価のものであった。
なお、３次ウイルス液５μｌを２４穴プレートの２９３細胞１ウエルに感染し、増殖したウイルスＤＮＡの酵素切断パターンを上記の方法で確認した。もし、欠失ウイルスあるいは親ウイルスとの混合物であることが疑われたら、２次ウイルス液の段階で既にわずかに混在していた欠失ウイルスが増殖が早いため見えてきた可能性があるので、全ての３次シードを廃棄して、別の２次ウイルス液から改めてやり直すか、その１次ウイルス液から限界希釈法により、目的のウイルスを純化した。
【０１２８】
【発明の効果】
本発明により、広範な動物細胞に外来遺伝子を安定な形で導入することのできる組換えＤＮＡウイルスを提供することができる。また、本発明はこの組換えＤＮＡウイルスの簡易な製造方法を提供する。特に、本発明の組換えアデノウイルスは遺伝病の治療に有用である。
【０１２９】
【配列表】

【０１３０】

【０１３１】

【０１３２】

【０１３３】

【０１３４】

【０１３５】

【０１３６】

【０１３７】

【０１３８】

【０１３９】

【０１４０】

【図面の簡単な説明】
【図１】図１は、コスミドｐＡｄｅｘ１ｃｗの構造を示す概念図である。
【図２】図２は、コスミドｐＡｄｅｘ１ＣＡｗｔの構造を示す概念図である。
【図３】図３は、プラスミドｐＡ６０Ｘ９９の構造を示す概念図である。
【図４】図４は、プラスミドｐＡ２Ｌ６０Ｘ９９の構造を示す概念図である。
【図５】図５は、プラスミドｐＡ２Ｌ３Ｌ６０９９の構造を示す概念図である。
【図６】図６は、組換えアデノウイルスＡｄｅｘ２Ｌ３ＬＣＡＮＬａｃＺおよびＡｄｅｘ１ＣＡＮＣｒｅを２９３細胞に共感染させた後に回収した細胞からＤＮＡを抽出し、これをテンプレートとしてＰＣＲを行った結果を示す図である。レーン１はプライマー（１）と（４）を用いた場合、レーン２はプライマー（２）と（３）を用いた場合の電気泳動図をそれぞれ示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a recombinant DNA virus for animal cell infection and a method for producing the same. Furthermore, the present invention relates to a recombinant DNA virus vector containing a DNA sequence encoding a recombinase recognition sequence used for the production of the recombinant DNA virus.
[0002]
[Prior art / problems to be solved by the invention]
In the past, retroviruses have been often used as viral vectors for gene transfer, but this virus can be introduced only into dividing cells and integrated into the host cell chromosomes, making it particularly safe for gene therapy. There is a problem from the viewpoint of safety and its application range is considered to be limited.
Adenovirus vectors have the advantage of showing nearly 100% introduction efficiency in various animal cultured cells, not having a mechanism of positive chromosome integration unlike retroviruses, and being able to introduce genes even in resting cells. The application range as a vector for exogenous gene transfer experiments is extremely wide, and it is thought that it will be established as one of the main technologies of gene therapy in the near future.
[0003]
The use of adenovirus vectors is rapidly spreading as one of gene therapy techniques and in terms of expression studies in highly differentiated cells such as the nervous system. As a gene therapy technique, research has been energetically promoted as a so-called in vivo gene therapy method that directly compensates for a deficient gene in living cells that have a function by directly administering it to an already constructed and functioning tissue. ing. As for cystic fibrosis, five groups in the United States have already received experimental treatment for patients, and they are actively researching for muscular dystrophy, familial hypercholesterolemia, brain tumors, etc. . On the other hand, adenoviral vectors can also introduce genes into resting cells, and primary cultures and experiments on gene introduction into individual animals have attracted attention as methods for introducing genes into differentiated cells, particularly the nervous system. Based on the above, adenovirus vectors can be used not only for gene therapy introduction into many differentiated and undifferentiated cells including the nervous system, but also for gene expression by direct injection / administration to individual animals. The application of is expected.
[0004]
However, unlike retroviruses, adenoviruses do not have an active mechanism of chromosomal integration, so expression is transient. The period is from 1 to 2 weeks and at most about 2 months. Therefore, when it is necessary to continue the therapeutic effect, it is desirable that the adenovirus is stably present in the cell for as long as possible, and continues to produce the expression product of the foreign gene inserted into the adenovirus. Recently, it has been found that the presence of the E2A gene region of the adenovirus genome has an adverse effect on the intracellular stability of adenovirus.
Accordingly, an object of the present invention is to construct a recombinant adenoviral vector system in which an adenoviral vector introduced into an animal cell exists more stably in the cell and can continue to produce a foreign gene product. Furthermore, it is to provide such a system for gene therapy.
[0005]
[Means for Solving the Problems]
Therefore, the present inventors have intensively studied to solve the above problems, and have found that a region into which foreign nucleotides can be introduced exists between the adenovirus E2A gene region and the L3 gene region. A foreign nucleotide can be directly introduced into this region by a conventional method, but a foreign linker may be introduced after a necessary restriction enzyme site is introduced once by introducing a suitable linker. The foreign nucleotide may be a foreign gene that encodes a polypeptide that is desired to be expressed in an animal cell after infection, and the foreign nucleotide directly serves as a substrate for an enzyme that coexists in the cell. There may be.
Furthermore, the present inventors introduced a recombinase recognition sequence as an exogenous nucleotide into the above region so that it can be used in combination with a recombinant DNA virus vector for infection of animal cells into which a recombinase gene that can be expressed in animal cells is inserted. It was found that an adenovirus vector system capable of deleting the E2A gene region of the adenovirus genome in animal cells can be constructed as a DNA virus vector for animal cell infection.
[0006]
Here, recombinase is a specific DNA recombination enzyme that recognizes a specific DNA sequence consisting of several tens of bases, and performs the entire process of DNA cutting, strand exchange and binding between these sequences. Therefore, when a recombinant adenoviral vector expressing this enzyme and a recombinant adenoviral vector having two copies of this recognition sequence in the same direction on both sides of the E2A gene region are prepared and co-infected with both cells, The recombinase causes a rearrangement between two recognition sequences, and the sandwiched portion is cut out as a circular molecule. Therefore, it can be expected that the adenoviral vector lacking the E2A gene region thus obtained is significantly more stable than the adenoviral vector containing the E2A gene region, and can be used advantageously for gene therapy.
[0007]
However, conventionally, it has been known that a foreign DNA sequence can be introduced into the right side of the E2A gene region (see FIG. 1), that is, into the E3 region. It was not known. The present inventors have now found that there is a site where a foreign DNA sequence can be introduced between the stop codon of the E2A gene and the stop codon of the L3 gene. Thus, it was revealed for the first time that a recombinase recognition sequence can be introduced across the E2A gene region while maintaining the function of adenovirus growth without impairing the function of the E2A gene.
The present invention has been completed by further research based on this knowledge.
[0008]
  That is, the gist of the present invention is that between the stop codon of the E2A gene and the stop codon of the L3 gene,Recombinase recognition sequenceThe present invention relates to a recombinant adenovirus for animal cell infection, characterized in that is inserted.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
The present invention is described in detail below.
The DNA virus vector for animal cell infection in the present invention can be used without particular limitation as long as it is a vector derived from a DNA virus that can exist only outside the chromosome after infection in the cell, such as adenovirus. it can. For example, an adenovirus vector, a vaccinia virus vector, a papopavirus vector, etc. are mentioned. Hereinafter, the present invention will be described using an adenovirus vector as a suitable example of a DNA virus vector for animal cell infection having a recombinase gene or a recombinase recognition sequence.
[0010]
The adenovirus used in the present invention has an animal as a natural host, and a human adenovirus having a human as a host is particularly preferably used. The genome of a human adenovirus is a double-stranded linear DNA of about 36 kbp, which has an inverted repeat base sequence consisting of approximately 100 bp at both ends of the DNA strand, and an E2B gene product at the 5 ′ end of both ends of the DNA strand. It has a unique structure in which the cut 55k protein is covalently bonded.
[0011]
The adenovirus genome used in the present invention preferably lacks the E1 gene region, particularly the E1A gene region. This is because the E1A gene region involved in adenovirus cell canceration activity is deleted, thereby detoxifying adenovirus and expressing only foreign gene sequences incorporated into the genome. Although it is not always necessary to delete the entire E1A gene region, the object can be achieved by removing, for example, 1.3 to 9.3% of fragments.
Further, the adenovirus genome used in the present invention may be deleted from the E3 gene region. In particular, those obtained by deleting 79.6-84.8% of the E3 gene region are preferable. This is because it is unnecessary for adenovirus replication.
Therefore, it has the feature that it cannot grow in host cells except for human embryonic kidney-derived cell lines (293 cells) that continuously express E1A and E1B genes.
[0012]
However, when actually administered to humans or animals, a protein having the same function as the E1A protein is present in the cells, and thus the adenovirus protein is slightly expressed. This has been shown to cause cellular immunity, and cells carrying viral DNA are attacked and eliminated. This is why the gene expression by the currently used E1A and E1B deficient adenovirus vectors is short-lived. In order to prevent this, it has been clarified by Yang et al. That the deletion of the function of the E2A gene is effective (Nature Genetics, vol. 7, 362-369, 1993). This uses a temperature-sensitive E2A gene mutant, but when administered to an animal, the functional expression of the E2A gene is suppressed, but the functional expression cannot be completely stopped. As a means of completely stopping the functional expression of the E2A gene, it is conceivable to delete the E2A gene region. However, since the E2A gene product is essential for the replication of the adenovirus genome, the adenovirus itself lacking the E2A gene It cannot grow even in 293 cells.
The present invention co-infects an animal culture cell with a recombinant adenovirus in which recombinase recognition sequences are arranged at both ends of the E2A gene and an adenovirus for recombinase expression, and the E2A gene-deficient infectious virus particle is expressed by the recombinase expressed in the cell. Is to produce. A sufficient amount of the E2A gene product is supplemented at least from an adenovirus for recombinase expression. Since the obtained E2A gene-deficient virus has no expression of the E2A gene, there is no doubt that the expression period of the target gene is greatly extended.
[0013]
The recombinase recognition sequence is inserted at the right side of the E2A gene region on the right side, but the left side of the E2A gene region is between the E2A gene stop codon and the L3 gene stop codon. Thus, a site that does not inhibit the growth of the resulting recombinant adenovirus is selected. If the E2A gene region or the L3 gene region is partially deleted or the poly A addition signal region is partially deleted, the resulting recombinant adenovirus is inhibited from growing, which is not preferable.
[0014]
Examples of promoters used in the present invention include animal virus gene promoters and animal cell gene promoters. Examples of the former include SV40 gene promoter, adenovirus major late gene promoter, and the like. Examples of the latter include thymidine kinase gene promoter, metallothionein gene promoter, immunoglobulin gene promoter, and the like. However, the CAG promoter is particularly advantageously used in the present invention. This promoter is a hybrid promoter comprising a cytomegalovirus enhancer, a chicken β-actin promoter, a rabbit β globin splicing acceptor and a poly β sequence derived from rabbit β globin. It is disclosed. The preparation is carried out by cutting out with the restriction enzymes SalI and HindIII from pCAGGS described in the publication (JP-A-3-16887, page 13, line 20 to page 20, line 14 and page 22, line 1 to page 25, line 6). Can be used in the present invention.
[0015]
The recombinase used in the present invention is a specific DNA recombination enzyme, which recognizes a specific base sequence and performs the entire process of DNA cleavage, strand exchange and binding between the sequences. Such enzymes include those encoded by E. coli bacteriophage P1 (recombinase Cre). This is the case with loxP in bacteriophage P1 (Abremski et al., J. Biol. Chem. 1984, 1509-1514; and Hoess et al., P.N.A.S., 1984,81, 1026-1029) The sequence is used as a substrate. That is, the loxP sequence is a recognition sequence for recombinase Cre. Other recombinases include recombinases encoded by the FLP gene derived from the yeast 2μ plasmid (James R. Broarch et al., Cell,29227-234). Furthermore, the thing derived from the pSR1 plasmid of Tigosaccharomyces louii can also be used. It is encoded by the R gene (Matsuzaki et al., Molecular and Cellular Biology,8955-962 (1988)). Among these, the recombinase of bacteriophage P1 is particularly suitable for the present invention.
[0016]
In the case of the recombinase gene, for example, in the case of the recombinase Cre gene, the portion encoding the recombinase gene of the DNA of bacteriophage P1 can be amplified using the polymerase chain reaction (PCR) method and used in the present invention. Other recombinase genes can be similarly prepared using the PCR method. The primer used in this case is selected so that the entire sequence of the recombinase gene is covered, and for the convenience of construction of the recombinant adenovirus vector, an appropriate restriction enzyme cleavage sequence is added to the outside of each primer. Is preferably used.
[0017]
The recombinase recognition sequence (substrate sequence) is several tens of bp, for example, the loxP sequence is 34 bp, and the base sequences are all known (Abremski et al., J. Biol. Chem. 1984, 1509). -1514; and Hoess et al., PNAS, 1984,81, 1026-1029), which can be used in the present invention after being chemically synthesized by a conventional method.
[0018]
The poly A sequence used in the present invention is not particularly limited, but those derived from rabbit β globin are particularly preferred.
[0019]
In the present invention, when the recombinase gene is incorporated into an adenovirus vector, it is preferable to incorporate a nuclear translocation signal sequence at the same time. For example, the nuclear translocation signal of SV40 can be used. This is because the recombinase expressed in the cytoplasm of the infected cell by the adenovirus vector needs to move into the nucleus in order to act on the adenovirus vector having the recognition sequence, and the nuclear translocation signal sequence promotes this ( Daniel Kalderon et al., Cell.39499-509 (1984)).
[0020]
The foreign gene used in the present invention is not particularly limited as long as it is a gene that can be expressed by the above-mentioned hybrid promoter (CAG promoter) or other promoters. Normal gene sequence corresponding to the defective gene (for example, adenosine deaminase, dystrophin, low density lipoprotein receptor, α-1 antitrypsin, blood coagulation factor 8, blood coagulation factor 9, galactosidase α or β), cytokines ( For example, interleukin-1-12, interferon-α, β or γ, tumor necrosis factor-α or β, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, erythropoietin, growth hormone, insulin, insulin-like growth hormone), Nerve Nutritional factors, non-self antigen genes (eg, allo HLA (HLA-B7)), nucleotide sequences encoding viral antigens, etc., tumor suppressor genes (eg, p53, RB, WT-1, NM23, NF-1), cancer Examples include antisense sequences such as Ras, which are genes, or suicide genes such as thymidine kinase and cytosine deaminase.
[0021]
The promoter, foreign gene and poly A sequence incorporated in the recombinant adenovirus vector of the present invention may be oriented in this order from the upstream or in the reverse order.
[0022]
Next, a method for producing the recombinant adenovirus of the present invention will be described.
(1) First, a method for producing a recombinant adenoviral vector having two recombinase recognition sequences facing in the same direction on both sides of the E2A gene region, a promoter, a foreign gene, and a poly A sequence will be described. For convenience, recombinase Cre is used as the recombinase, loxP is used as the recognition sequence, the CAG promoter is used as the promoter and polyA sequence, and the LacZ gene is used as the foreign gene. A substantially similar approach can be utilized when using sequences, promoters and poly A sequences.
[0023]
(A) (Construction of pAdexlCAwt)
(1) Construction of plasmid pCMwCH31 containing CAG promoter
A plasmid pCAGGS (Niwa et al., Gene, 108, 193-200 (1990)) containing a CAG promoter is digested with EcoRI, then blunted with Klenow enzyme, and subjected to a ligase reaction with a SwaI linker. Next, the obtained plasmid is cleaved with SalI, smoothed with Klenow enzyme, and subjected to ligase reaction with ClaI linker. Further, the obtained plasmid is cleaved with PstI, smoothed with Klenow enzyme, and subjected to ligase reaction with XhoI linker to obtain plasmid pCMwCH31 containing CAG promoter.
The disappearance of the original restriction enzyme site and the insertion of each linker are confirmed by agarose gel electrophoresis after each restriction enzyme cleavage.
[0024]
(2) Construction of pAdex1c
Example 1 (2) Constructed by the method described in (i) to (vi). The outline is as follows.
A plasmid (pUAF0-17D) containing 17% of the left end of the adenovirus genome from which the E1 gene region was deleted (pUAF0-17D), a fragment obtained by ligating a BamH linker to type 5 adenovirus DNA and then digesting with HindIII (2.8 kb, adeno A plasmid (pUAF0-8) obtained by inserting into pUC19 (equivalent to 8% of the left end of the viral genome), and a 3.4 kb fragment obtained by digesting adenoviral DNA with HindIII (8- 17%) is inserted into the HindIII site of pUC19 to prepare a plasmid (pUAF8-17), followed by a 454 bp BamHI-ClaI fragment derived from pUAF0-8 and a 2.9 kb HindIII derived from pUAF8-17. -ClaI hula And pUAF0-17D is obtained by inserting into the BamHI / HindIII site of pUC19.
[0025]
Furthermore, type 5 adenovirus DNA is digested with Bst1107 and EcoRI to obtain a 21.6 kb fragment. In addition, an EcoRI-SalI fragment (6.5 kb) of pX2W derived from the adenovirus genome is prepared.
On the other hand, charomid 9-11 (I. Saito & G. Stark, Proc. Natl. Acad. Sci. USA, vol. 83, p8664-8668, 1986) was digested with Asp718 and BamHI, blunted with Klenow enzyme, and self-treated. Ligate. A BamHI linker is then inserted into the EcoRI site to prepare the shalomid chdRBR7-11.
[0026]
The above-described BamHI-Bst1107 fragment (2.9 kb) of pUAF0-17D, Bst1107-EcoRI fragment (21.6 kb) of adenovirus genome and EcoRI-SwaI fragment (6.5 kb) of pX2W were digested with EcoRI and Ecl36I chdRBR7- 11 and ligate. Thereafter, it is packaged in vitro and infected with DH5α. A transformant having the desired fragment is isolated and named pAdex1c.
[0027]
(3) Construction of cassette cosmid pAdex1cw
After mixing with pAdex1c cleaved with ClaI and recovered by ethanol precipitation, the following synthetic linker (1) (SEQ ID NO: 2) subjected to 5′-terminal phosphorylation (including SwaI, ClaI, SalI and NruI sites) was mixed, Binding overnight in a reaction solution containing ATP, T4 DNA ligase. The ligase is heat-inactivated and then digested with SwaI. This cleavage is carried out in order to obtain a cosmid having a structure in which only one linker is inserted by cutting out a SwaI fragment from a plurality of linkers inserted. Next, the reaction solution is subjected to Spun column (manufactured by Pharmacia) to remove a small fragment derived from the linker, followed by ligation with T4 DNA ligase and circularization by self-annealing. Subsequently, in vitro packaging is performed, and the structure of cosmid DNA prepared from each colony is confirmed by simultaneous digestion with BamHI and NruI. When inserted in the target direction, a fragment of 483 bp is generated, and when inserted in the reverse direction, a fragment of 464 bp is generated. The target cassette cosmid pAdex1cw (FIG. 1) is obtained by this confirmation.
Synthetic linker

[0028]
(4) Construction of cassette cosmid pAdexlpCAw
The plasmid pCMwCH31 constructed in {circle around (1)} is co-digested with HindIII and ClaI, blunted with Klenow enzyme, and ligated with a PmeI linker subjected to 5 ′ end phosphorylation. The ligase is heat inactivated and then digested with Psp1406I. This cleavage is performed in order to obtain a fragment having a structure in which a Psp1406I fragment is excised from the one in which a plurality of linkers are inserted, and one linker is linked to each end of the DNA fragment. Thereafter, the reaction solution is subjected to agarose gel electrophoresis, a gel containing a 2.3 kb DNA fragment is cut out, and the DNA fragment is recovered from the gel by electrophoresis. Next, pAdexlcw is cleaved with ClaI, and then the resulting small fragment is removed with Spun column (Pharmacia) and the aforementioned 2.3 kb DNA fragment is ligated with T4 DNA ligase. After heat inactivating the ligase, ClaI is added to cleave the cyclic cosmid generated by self-annealing. Then used for in vitro packaging.
Furthermore, the structure of cosmid DNA prepared from each colony is confirmed by simultaneous digestion with BamHI and XhoI. When inserted in the target direction, 483 bp and 4.8 kb are generated, and when inserted in the reverse direction, 2.7 and 2.5 kb fragments are generated. The target cassette cosmid pAdexlpCAw is obtained by this confirmation.
[0029]
(5) Construction of cassette cosmid pAdexlCAwt (Cell engineering, Vol.13, No.8, P759)
The following synthetic linker (2) (SEQ ID NO: 3) obtained by cleaving with SwaI and then recovered by ethanol precipitation and subjected to 5′-terminal phosphorylation (including ClaI, XbaI, SpeI, PacI, SwaI and ClaI sites) Are mixed together overnight in a reaction solution containing ATP and T4 DNA ligase. After heat-inactivating the ligase, PacI (20 units) is added and reacted. This cleavage is carried out in order to obtain a cosmid having a structure in which only one linker is inserted by cutting out a PacI fragment from a plurality of linkers inserted. Next, the reaction solution is subjected to Spun column (manufactured by Pharmacia) to remove a small fragment derived from the linker, and then allowed to bind overnight in a reaction solution containing T4 DNA ligase, followed by circularization by self-annealing. Ligase is heat-inactivated and used for in vitro packaging. Next, the structure of cosmid DNA prepared from each colony is confirmed by simultaneous digestion with XbaI and XhoI. When inserted in the target direction, a fragment of 552 bp is generated, and when inserted in the reverse direction, a fragment of 568 bp is generated. By confirming this, the target cassette cosmid pAdexlCAwt (FIG. 2) is obtained.
Synthetic linker structure

[0030]
(B) (Construction of loxP-inserted cosmid 1)
Preparation of pAdex2L3LCAwt
(1) Preparation of pA60X99X
After cutting pAdexlCAwt with BamHI, BamHI is deactivated by heat treatment. Then, bind overnight with T4 DNA ligase. Subsequently, Escherichia coli DH5α (manufactured by GIBCO BRL) is transformed using this reaction mixture, and plasmid DNA is prepared from the obtained transformant to obtain the target plasmid pA60X99X.
[0031]
(2) Construction of pA60X99 (removal of XbaI site other than adenovirus)
pA60X99X was treated with XbaI, the reaction mixture was subjected to agarose gel electrophoresis, a gel containing a 23.8 kb DNA fragment cleaved at only one of the two XbaI sites was excised, and the DNA fragment was recovered from the gel by electrophoresis To do. Next, this fragment is blunted at both ends with Klenow enzyme (Takara Shuzo) and ligated overnight with T4 DNA ligase. Next, E. coli DH5α is transformed using this reaction mixture, and plasmid DNA is prepared from the obtained transformant. These plasmid DNAs are co-digested with BsrGI and XbaI to obtain a 6.2 kb fragment, plasmid pA60X99 (FIG. 3).
[0032]
(3) Construction of pA2L60X99 (insertion of loxP into BsrGI site)
After cutting pULL2r with Xhol, both ends are smoothed with Klenow enzyme (Takara Shuzo). Thereafter, phenol: chloroform (1: 1) treatment is performed, followed by ethanol precipitation. The precipitate is obtained by centrifugation and dissolved in 60 μl TE. This is ligated overnight in a ligase reaction solution (final volume 50 μl) containing 5′-terminal phosphorylated KpnI linker (Takara Shuzo), ATP and T4 DNA ligase. Next, it digests with Asp718 (made by Boehringer). The reaction mixture is subjected to agarose gel electrophoresis, a gel containing a 64 bp DNA fragment containing loxP is cut out, and the DNA fragment is recovered from the gel by electrophoresis.
[0033]
In addition, said pULL2r is prepared as follows. pUC119 (manufactured by Takara Shuzo) was cleaved with the restriction enzyme Ecl136II, treated with alkaline phosphatase, and then the MloI site and the XhoI site at the ends, which were combined with each other and contained a loxP sequence designed to generate an NruI site. A ligation reaction with the synthetic DNA fragment (SEQ ID NO: 4) is performed to obtain a plasmid pULL2r in which two of the synthetic DNA fragments are inserted.

(The underlined sequence is the loxP site.)
[0034]
On the other hand, plasmid pA60X99 (10 μg) was cleaved in 50 μl of a reaction system containing BsrGI (50 units), the reaction mixture was subjected to agarose gel electrophoresis, a gel containing a 23.8 kb DNA fragment was excised, and DNA was extracted from the gel by electrophoresis. Collect the fragments. This DNA fragment is allowed to bind overnight in a ligase reaction solution containing a 64 bp DNA fragment containing loxP, ATP and T4 DNA ligase. Sterile water and a BsrGI reaction buffer are added thereto, and the ligase is heat-inactivated by incubating at 70 ° C. for 10 minutes, and then treated with BsrGI to cleave the cyclic pA60X99 produced by self-annealing. Next, 10 μl of this reaction mixture is used to transform E. coli DH5α, and plasmid DNA is prepared from the resulting transformant.
[0035]
In order to confirm the direction of the inserted loxP, ApaI and MluI are simultaneously digested, and the reaction mixture is subjected to agarose gel electrophoresis. When inserted in the intended direction, 366 and 219 bp fragments are generated, and when inserted in the reverse direction, 334 and 251 bp fragments are generated. In addition, when digested with NruI, a fragment of 573 bp is generated when inserted in the intended direction, and a fragment of 533 bp is generated when inserted in the reverse direction. Furthermore, when co-digested with DraI and KpnI, a fragment of 384 bp is generated when one loxP is inserted in the target direction and 320 bp is inserted when two are inserted. A target plasmid pA2L60X99 (FIG. 4) in which all these three conditions are satisfied, that is, one loxP is inserted in the target direction is obtained.
[0036]
(4) Construction of pA2L3L6099 (insertion of loxP into XbaI site)
After cleaving pULL2r in 100 μl of a reaction system containing XhoI, both ends are smoothed with Klenow enzyme (Takara Shuzo). Subsequently, after phenol: chloroform (1: 1) treatment, ethanol precipitation is performed. The precipitate is obtained by centrifugation and dissolved in TE. This is ligated overnight in a reaction solution containing a 5'-terminal phosphorylated SpeI linker (Takara Shuzo), ATP and T4 DNA ligase. Furthermore, after digesting with SpeI, the reaction mixture is subjected to agarose gel electrophoresis, a gel containing a 64 bp DNA fragment containing loxP is excised, and the DNA fragment is recovered from the gel by electrophoresis.
[0037]
On the other hand, after cutting pA2L60X99 with XbaI, the reaction mixture is subjected to agarose gel electrophoresis, a gel containing a 23.8 kb DNA fragment is cut out, and DNA is recovered from the gel by electrophoresis. This DNA fragment is bound overnight in a reaction solution containing a 64 bp DNA fragment containing loxP, ATP and T4 DNA ligase. The ligase is heat-inactivated and then treated with XbaI to cleave the cyclic pA2L60X99 produced by self-annealing. Using this reaction mixture, E. coli DH5α is transformed, and plasmid DNA is prepared from the resulting transformant.
[0038]
In order to confirm the direction of the inserted loxP, BglII and MluI are simultaneously digested, and the reaction mixture is subjected to agarose gel electrophoresis. When inserted in the intended direction, 366 and 503 bp fragments are generated, and when inserted in the reverse direction, 398 and 471 bp fragments are generated. When ApaI and MluI are digested simultaneously, a fragment of 660 bp is generated when inserted in the target direction, and a fragment of 628 bp is generated when inserted in the reverse direction. When digested with EcoNI and MluI, a fragment of 311 bp is generated when inserted in the target direction and 343 bp when inserted in the reverse direction. Further, when digested simultaneously with HpaI and SacI, a fragment of 397 bp is generated when one loxP is inserted in the target direction, and a 461 bp fragment is generated when two are inserted. A target plasmid pA2L3L6099 (FIG. 5) in which all these four conditions are satisfied, that is, one loxP is inserted in the target direction is obtained.
[0039]
(5) Production of pAdex2L3LCAwt
pAdex1CAwt is cleaved with Csp45I (manufactured by Toyobo), then cleaved with BamHI and EcoRI in the same reaction solution, and then checked by agarose gel electrophoresis. A gel containing a 21 kb DNA fragment is cut out, and the DNA fragment is recovered from the gel by electrophoresis. The cleavage with Csp451 and EcoRI is for preventing contamination of other fragments when recovering the 21 kb BamHI DNA fragment.
[0040]
After cutting pA2L3L6099 with BamHI, it is treated with phenol: chloroform (1: 1), and the aqueous layer is subjected to gel filtration with Sephadex G25 equilibrated with TE. The recovered DNA fragment is bound overnight in a reaction solution containing the 21 kb DNA fragment, ATP and T4 DNA ligase. The ligase is heat inactivated and then used for in vitro packaging.
[0041]
That is, Gigaback XL (manufactured by Stratagene), which is a lambda in vitro packaging kit, is used, and the rest is frozen at -80 ° C. Since GIGABACK XL has a low packaging efficiency for cosmids of 42 kb or less, it can be selected to some extent from cosmids that have grown with inserts. In the present invention, if 10 colonies are picked up, most of them contain an insert, and the target clone (a clone in which the virus genome is correctly linked) can be easily obtained. The cosmid is handled according to the conventional method (Izumi Saito et al., Experimental Medicine: 7: 183-187, 1989).
[0042]
The packaged cosmid is infected with E. coli DH5α (GibcoBRL). That is, Ap⁺(Ampicillin added) Agar plate and Ap⁺LB (pool) is inoculated at various concentrations and cultured overnight. Pool miniprep DNA is extracted and prepared, and the ratio of inserts inserted by digestion with restriction enzyme DraI is examined. Take the whole colony with agar⁺Incubate overnight in LB to prepare miniprep DNA. Next, the structure of cosmid DNA prepared from each colony is confirmed by DraI digestion. When inserted in the target direction, 891 bp is generated, and when inserted in the reverse direction, a 1.4 kb fragment is generated. Thereby, the target cassette cosmid pAdex2L3LCAwt is obtained.
[0043]
(C) (Construction of loxP-inserted cosmid 2)
Preparation of pAdex2LA3LCAwt
(1) Production of pUCA6065
pA60X99 is cleaved with BamHI and PstI, the reaction mixture is subjected to agarose gel electrophoresis, a gel containing a 1.7 kb DNA fragment containing the BsrGI site is excised, and the DNA fragment is recovered from the gel by electrophoresis. In the same manner, pUC19 is cleaved with BamHI and PstI to recover a 2.7 kb fragment. Next, both fragments are added to a ligase reaction buffer, and ATP and T4 DNA ligase are further added and allowed to bind overnight. The reaction mixture is then used to transform E. coli DH5α, and plasmid DNA is prepared from the resulting transformant to obtain the desired plasmid pUCA6065.
[0044]
(2) Production of p2LA6065
pUCA6065 is cut with BamHI and AflIII to prepare a 780 bp fragment, and the same plasmid is cut with BamHI and BsrGI to prepare a 3.6 kb fragment. These both fragments and the following linker DNA (SEQ ID NO: 11) containing the loxP sequence are mixed and added to the ligase reaction buffer, and further ATP and T4 DNA ligase are added and allowed to bind overnight. Next, using this reaction mixture, E. coli DH5α is transformed, plasmid DNA is prepared from the obtained transformant, and the target plasmid p2LA6065 into which one linker DNA has been inserted is obtained.

[0045]
(3) Preparation of pA2LA3L6099
p2LA6065 is cut with BamHI and SfiI (or BglI) to prepare a 1.5 kb fragment. PA2L3L6099 is cleaved with BamHI and SfiI to prepare a fragment of about 22 kb. Next, both fragments are added to a ligase reaction buffer, and ATP and T4 DNA ligase are further added and allowed to bind overnight. The reaction mixture is then used to transform E. coli DH5α, and plasmid DNA is prepared from the resulting transformant to obtain the desired plasmid pA2LA3L6099.
[0046]
(4) Preparation of pAdex2LA3LCAwt
pAdex2LA3LCAwt is prepared from pA2LA3L6099 and pAdexlCAwt by the same operation as in (5) in the production of pAdex2L3LCAwt.
[0047]
(D) (construction of loxP insertion cosmid 3)
Production of pAdex2LD3LCAwt
(1) Production of pHSGA6065
pA60X99 is cleaved with BamHI and PstI, the reaction mixture is subjected to agarose gel electrophoresis, a gel containing a 1.7 kb DNA fragment containing the BsrGI site is excised, and the DNA fragment is recovered from the gel by electrophoresis. pHSG299 (Takara Shuzo) is cleaved with BamHI and PstI, and a 2.7 kb fragment is recovered by the same operation. Next, both fragments are added to a ligase reaction buffer, and ATP and T4 DNA ligase are further added and allowed to bind overnight. The reaction mixture is then used to transform E. coli DH5α, and plasmid DNA is prepared from the resulting transformant to obtain the desired plasmid pHSGA6065.
[0048]
(2) Production of p2LD6065
pHSGA6065 was cut with BsrGI and DraI to prepare a 4.4 kb fragment, this was mixed with the following linker DNA (SEQ ID NO: 12) containing the loxP sequence, added to the ligase reaction buffer, and further ATP and T4 DNA ligase were added. Add and bind overnight. The reaction mixture is then used to transform E. coli DH5α, and plasmid DNA is prepared from the resulting transformant to obtain the desired plasmid p2LD6065 into which one linker DNA has been inserted.

[0049]
(3) Production of pA2LD3L6099
p2LD6065 is cleaved with BamHI and SfiI (or BglI) to prepare a 1.5 kb fragment. PA2L3L6099 is cleaved with BamHI and SfiI to prepare a fragment of about 22 kb. Next, both fragments are added to a ligase reaction buffer, and ATP and T4 DNA ligase are further added and allowed to bind overnight. Next, E. coli DH5α is transformed using this reaction mixture, and plasmid DNA is prepared from the resulting transformant to obtain the desired plasmid pA2LD3L6099.
[0050]
(4) Preparation of pAdex2LD3LCAwt
The pAdex2LD3LCAwt is produced from pA2LD3L6099 and pAdexlCAwt by the same operation as in (5) in the production of pAdex2L3LCAwt.
[0051]
(E) Preparation of adenovirus DNA-terminal protein complexes (Ad5 dlX DNA-TPC and Adex1CANLacZ DNA-TPC)
(1) As adenovirus DNA, Ad5 dlX (I. Saito et al., J. Virology, vol. 54, 711-719 (1985)) or Adex1CANLacZ is used. Ad5 dlX is infected with HeLa cells (for 10 Roux) and dex1CANLacZ is infected with 293 cells, respectively, and cultured.
That is, a virus solution of Ad5-dlX or Adex1CANLacZ (-10⁹PFU / ml) is infected with 0.2 ml / Roux, and after 3 days, detached cells are collected by centrifugation. Most of the adenovirus particles are in the cell nucleus, not in the medium, which has the advantage that the virus can be purified from infected cells. (The following operations are performed aseptically.)
[0052]
{Circle around (2)} The obtained cells are suspended in Tris-HCl (pH 8.0), the cells are disrupted using a sealed sonicator, and the virus is released from the cells.
(3) After removing the precipitate from the resulting crushed material by centrifugation, put the cesium chloride solution (specific gravity 1.43) into the ultracentrifuge SW28 tube, overlay the supernatant on it, and concentrate by cushion centrifugation I do.
(4) Transfer the virus layer just below the interface to the SW50.1 tube. The virus layer just below the interface is usually visible, and 5 ml of the virus layer and the cesium chloride below it are collected. At the same time, the other is filled with a cesium chloride solution (specific gravity 1.34).
These were ultracentrifuged overnight at 35 krpm and 4 ° C. Next, the white virus band is separated and transferred to a tube with a gradient. Furthermore, it is subjected to ultracentrifugation at 35 krpm, 4 ° C. for 4 hours or more.
[0053]
(5) Separate a white virus band, mix with an equal amount of 8M guanidine hydrochloride at room temperature, add 4M guanidine saturated cesium chloride and fill into a VTi65 tube. With 4M guanidine hydrochloride, the particle protein is denatured and dissociated, and DNA-TPC is released.
[0054]
(6) The above tube was ultracentrifuged at 55 krpm and 15 ° C. overnight, fractionated by 0.2 ml, 1 μl of each was mixed with 20 μl of 1 μg / ml ethidium bromide aqueous solution, and the DNA was stained by fluorescence staining. Check for presence. Collect 2-3 fractions containing DNA.
(7) Dialyzed overnight (twice) in 500 ml of TE and stored at -80 ° C. The amount of Ad5dlX DNA-TPC or dex1CANLacZ DNA-TPC thus obtained is OD₂₆₀To calculate in the same manner as normal DNA.
(8) The resulting Ad5dlX DNA-TPC or Adex1CANLacZDNA-TPC was cleaved with a sufficient amount of AgeI for 2 hours to prepare a loxP-inserted recombinant adenovirus in the next step, and subjected to gel filtration with a Sephadex G25 column. Store at ℃.
[0055]
(F) Production of loxP-inserted recombinant adenovirus
NLacZ is obtained by adding a nuclear translocation signal of SV40 to the N-terminus of the E. coli LacZ gene.
{Circle around (1)} Prepare one each of 6cm and 10cm dishes of 293 cells cultured in DME supplemented with 10% FCS.
(2) -1. (Production of Ad5dlX2L3L or Adex2L3LCANLacZ)
8 μg of the cosmid pAdex2L3LCAwt DNA into which loxP incorporating the expression unit was inserted and 1 μg of Ad5dlX DNA-TPC or AgeI-cleaved Adex1CANLacZ DNA-TPC cleaved with AgeI were mixed using a Cellfect (Pharmacia) kit. One sheet is transfected by the calcium phosphate method. The mixed solution is dropped from above the medium of 6 cm petri dish and the culture is continued.
Incubate overnight (about 16 hours), change the culture medium in the morning, and in the evening, add 3% collagen-coated 96-wells (stock solution, 10-fold dilution, 100-fold dilution) to each well using 5% FCS-added DME. Re-roll with 0.1 ml. In order to prevent the number of cells from greatly differing between each plate, 293 cells of 10 cm dishes are mixed and spread on the two dilutions.
[0056]
(2) -2. (Production of Ad5dlX2LA3L or Addex2LA3LCANLacZ)
8 μg of cosmid pAdex2LA3LCAwt DNA into which loxP incorporating an expression unit was inserted and 1 μg of Ad5dlX DNA-TPC or AgeI-cleaved Adex1CANLacZ DNA-TPC cleaved with AgeI were mixed using a Cellfect (Pharmacia) kit. One sheet is transfected by the calcium phosphate method. The mixed solution is dropped from above the medium of 6 cm petri dish and the culture is continued.
Incubate overnight (about 16 hours), change the culture medium in the morning, and in the evening, add 3% collagen-coated 96-wells (stock solution, 10-fold dilution, 100-fold dilution) to each well using 5% FCS-added DME. Re-roll with 0.1 ml. In order to prevent the number of cells from greatly differing between each plate, 293 cells of 10 cm dishes are mixed and spread on the two dilutions.
[0057]
(2) -3. (Production of Ad5dlX2LD3L or Addex2LD3LCANLacZ)
8 μg of cosmid pAdex2LD3LCAwt DNA into which loxP incorporating an expression unit was inserted and 1 μg of Ad5dlX DNA-TPC or AgeI cleaved with AgeI were mixed with 6 μl of Cellfect (Pharmacia) kit. One sheet is transfected by the calcium phosphate method. The mixed solution is dropped from above the medium of 6 cm petri dish and the culture is continued.
Incubate overnight (about 16 hours), change the culture medium in the morning, and in the evening, add 3% collagen-coated 96-wells (stock solution, 10-fold dilution, 100-fold dilution) to each well using 5% FCS-added DME. Re-roll with 0.1 ml. In order to prevent the number of cells from greatly differing between each plate, 293 cells of 10 cm dishes are mixed and spread on the two dilutions.
[0058]
(3) After 3 to 4 days and 8 to 10 days, add 50 μl of 10% FCS-added DME to each well. Add 293 cells as soon as they thin.
Wells in which the virus has grown and the cells have died appear between 7 and 20 days. Each time the cells in the well are completely killed, the culture solution (every dead cell) is aseptically transferred to a sterile 1.5 ml tube with a sterile Pasteur pipette, snap frozen with dry ice and stored at -80 ° C.
(4) The determination is completed in 15 to 25 days. About 10 culture solution tubes collected from wells where cells died relatively slowly are selected, and after sonication, the supernatant obtained by centrifugation at 5000 rpm for 10 minutes is stored at −80 ° C. as the first virus solution (first seed). . This is because a well in which virus propagation has occurred earlier is likely to be a mixed infection of a plurality of virus strains.
[0059]
(5) Prepare 293 cells in a 24-well plate, and add 2% each of 5% FCS-DME (0.4 ml / well) and primary virus solution (10 μl).
(6) When the cells are completely killed in about 3 days, a supernatant is obtained by sonication and centrifugation in the same manner as in the preparation of the primary virus solution, and this is used as a secondary virus solution (second seed) at -80. Store at ℃. The other 1-well dead cells are centrifuged at 5000 rpm for 5 minutes, the supernatant is discarded, and only the cells are stored at −80 ° C. (cell pack). When cell packs of 10 virus strains are collected, the total DNA of infected cells is extracted by the following method. To the cell pack, 400 μl of TNE for cell DNA (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA), 4 μl of proteinase K (10 mg / ml) and 4 μl of 10% SDS are added.
[0060]
(7) After treatment at 50 ° C. for 1 hour, the nucleic acid obtained by phenol / chloroform extraction twice, chloroform extraction twice, and ethanol precipitation is dissolved in 50 μl of TE containing 20 μg / ml of RNase. 15 μl of the expression unit was cleaved with XhoI, an enzyme containing CG as a recognition sequence among the enzymes that cleave the expression unit, and the electrophoresis was performed overnight on an agarose gel with a length of about 15 cm along with XhoI cleavage of the expression cosmid cassette. Compare patterns. Since XhoI has a recognition site in the inserted loxP sequence, a clone showing a cleavage pattern in which two loxPs are inserted is selected. Discard the clones where the unexplainable bands appear thin because they may be mixed with the virus with the deletion.
[0061]
Using the obtained loxP-inserted recombinant adenoviruses Ad5dlX2L3L, Ad5dlX2LA3L, Ad5dlX2LD3L, etc. and a cosmid containing the target foreign gene expression unit, a known recombinant adenovirus production method such as the COS-TPC method (“Experimental Medicine Separate Volume” The recombinant adenovirus in which the target foreign gene expression unit and loxP are inserted can be prepared according to BioManual Series 4, Gene Introduction and Expression / Analysis Method, pages 43 to 58).
[0062]
(G) Preparation and structure confirmation of E2A gene-deficient adenovirus
Recombinant adenoviruses Adex2L3LCANLacZ and Adex1CANCre are infected with 293 cells at moi = 10 and 3, respectively, and cultured. On day 4, cells are collected and DNA is prepared by the method described above. The generation of dexdl23CANLacZ having a structure in which the region sandwiched by loxP (including the E2A gene) is cut out is confirmed by two methods.
Note that the structure of dex2LA3LCANLacZ and dex2LD3LCANLacZ can be confirmed by the same operation.
[0063]
1. SmaI digestion
As a result of gel electrophoresis after SmaI digestion, a 4.7 kb fragment produced by cutting out the region sandwiched by loxP is observed. A comparison of the density of this band and the 4.45 kb band commonly found in Adex2L3LCANLacZ, Adex1CANCre, and Adexdl23CANLacZ reveals about what percentage of the recovered recombinant adenovirus is Adexdl23CANLacZ.
[0064]
2. Confirmation by PCR
PCR is performed under normal conditions using 0.1 ng of the prepared DNA as a template, and the product is analyzed by agarose gel electrophoresis. The primers used are the oligonucleotide (1) (SEQ ID NO: 5), oligonucleotide (2) (SEQ ID NO: 6), oligonucleotide (3) (SEQ ID NO: 7) and oligonucleotide (4) (sequence) shown below. Number: 8) is preferred.

The PCR reaction solution composition was 50 mM KCl, 1.5 mM MgCl in 10 mM Tris.HCl (pH 8.3).₂, 0.2 mM dNTP mixture and each 0.2 μM primer, 0.1 ng template DNA and 0.5 unit Taq polymerase are preferred.
As a preferable example of PCR reaction conditions, double-strand dissociation is 1.5 minutes at 95 ° C., annealing is 1.0 minute at 64 ° C., extension reaction is 1.0 minute at 70 ° C., and 30 reaction cycles. is there.
When (1) and (4) were used as primers, a band estimated to be 393 bp was detected, revealing the presence of dexdl23CANLacZ from which the E2A gene was deleted, and when using (2) and (3) A band estimated to be 221 bp was detected, supporting the presence of the circular E2A gene excised by the Cre gene product, and the generation of dexdl23CANLacZ in which the region (including E2A gene) cleaved by loxP was excised from Adex2L3LCANLacZ It becomes clear (FIG. 6).
[0065]
(2) Next, a method for producing a recombinant adenovirus vector having a promoter, a recombinase gene and a poly A sequence will be described.
Hereinafter, the case where the recombinase Cre gene is used as the recombinase gene will be described, but the same applies to other recombinase genes.
[0066]
(1) Recombinase Cre gene prepared by PCR and plasmid pUC19 (Takara Shuzo) were digested with restriction enzymes PstI (Takara Shuzo) and XbaI (Takara Shuzo), respectively, mixed and ligated to incorporate the recombinase Cre gene. Plasmid pUCCre is obtained.
(2) Cassette cosmid pAdex1CAwt containing CAG promoter was treated with restriction enzyme SwaI (Boehringer) and pUCCre was digested with restriction enzymes PstI (Takara Shuzo) and XbaI (Takara Shuzo) and then Klenow enzyme (Takara Shuzo) And mix with smoothed ends. Next, the cassette cosmid is precipitated and ligated with T4 DNA ligase to obtain a cassette cosmid incorporating the recombinase Cre gene.
[0067]
When using a promoter other than the CAG promoter, first, the E3 region (1.9 kb) and the E1A / E1B region (2.9 kb) unnecessary for replication were deleted from the full length of the adenovirus genome (36 kb). A cassette cosmid having a genomic DNA of about 31 kb was prepared. On the other hand, a plasmid containing a promoter to be used, a recombinase Cre gene, and a poly A sequence was prepared and treated with an appropriate restriction enzyme, and E1A / E1B of the adenovirus genome was prepared. A cassette cosmid incorporating a recombinase Cre gene expression unit at the deletion site is obtained.
(3) Next, the obtained cassette cosmid is subjected to in vitro packaging using Gigapack XL (manufactured by Stratagene), which is a lambda in vitro packaging kit.
[0068]
(4) On the other hand, an adenovirus DNA-terminal protein complex (Ad5dlX DNA-TPC) is prepared. As adenovirus DNA, Ad5dlX (I. Saito et al., J. Virology, vol.54, 711-719 (1985)) was used, and Ad5dlX was infected to HeLa cells (for 10 Roux bottles). Do. Viral particles are collected, and DNA-TPC is separated and collected by treatment with guanidine hydrochloride and ultracentrifugation.
The Ad5dlX DNA-TPC thus obtained is treated with a sufficient amount of EcoT22I for the production of the next step of recombinant adenovirus.
[0069]
(5) As the final step, a cassette cosmid incorporating the recombinase Cre gene and Ad5dlX DNA-TPC treated with EcoT22I are mixed, and transfection is carried out by the calcium phosphate method using a Cellfect (Pharmacia) kit. A virus solution is collected from a cell that has been killed due to virus growth to obtain a recombinant adenovirus vector having a promoter, a recombinase gene, and a poly A sequence.
[0070]
The high-titer virus solution of the recombinant adenovirus of the present invention having the target foreign gene expression unit obtained as described above by the method of the present invention and completely lacking the function of the E2A gene is appropriately diluted. And co-infected by local injection (central nervous system, portal vein, etc.), oral (using enteric solvent) administration, respiratory tract administration, transdermal administration, etc., and used for treatment of various diseases including genetic diseases Can do.
[0071]
【Example】
EXAMPLES Hereinafter, although an Example and a reference example demonstrate this invention further in detail, this invention is not limited at all by these Examples.
Unless otherwise specified, various procedures for handling phages, plasmids, DNA, various enzymes, Escherichia coli, cultured cells, etc. in the Examples are described in “Molecular Cloning, A Laboratory Manual, edited by T. Maniatis et al., Second Edition (1989). ), Cold Spring Harbor Laboratory ". DNA restriction enzymes and modification enzymes were purchased from Takara Shuzo, New England Biolabs (NEB), Stratagene, or Boehringer, and used according to the manufacturer's instructions.
[0072]
Example 1 (construction of pAdex1CAwt)
(1) Construction of plasmid pCMwCH31 containing CAG promoter
A plasmid pCAGGS (Niwa et al., Gene, 108, 193-200 (1990)) containing a CAG promoter was digested with EcoRI, then blunted with Klenow enzyme, and subjected to a ligase reaction with a SwaI linker. Next, the obtained plasmid was cleaved with SalI, smoothed with Klenow enzyme, and ligase reaction with ClaI linker was performed. Further, the obtained plasmid was cleaved with PstI, then smoothed with Klenow enzyme, and ligase reaction with XhoI linker was performed. The disappearance of the original restriction enzyme site and the insertion of each linker were confirmed by agarose gel electrophoresis after each restriction enzyme cleavage.
[0073]
(2) Construction of pAdex1c
(I) Preparation of a plasmid (pUAF0-17D) containing 17% of the left end of the adenovirus genome lacking the E1 gene region
The type 5 adenovirus DNA was treated with S1 to make it blunt, and a BamH linker was ligated to the blunt end, followed by HindIII digestion, and the desired fragment (2.8 kb, corresponding to 8% of the left end of the adenovirus genome) was agarose. It was separated and recovered by gel electrophoresis and inserted into the BamHI / HindIII site of pUC19 digested with BamHI / HindIII. The obtained plasmid of interest was named pUAF0-8.
[0074]
(Ii) Adenovirus DNA was digested with HindIII, separated by agarose gel electrophoresis, the desired 3.4 kb fragment (corresponding to 8-17% of the left end of the adenovirus genome) was recovered from the gel, and the HindIII site of pUC19 (Named pUAF8-17).
The base number of pUAF0-8 (the base number here is derived from adenovirus DNA) The 454th PvuII site was converted to a ClaI site using a ClaI linker. This plasmid was digested with BamHI / ClaI, and a 454 bp BamHI-ClaI fragment was recovered by agarose gel electrophoresis.
The BglII site at base number 3328 of pUAF8-17 was converted to a ClaI site using a ClaI linker. This plasmid was digested with HindIII / ClaI, and a 2.9 kb HindIII-ClaI fragment was recovered by agarose gel electrophoresis.
The 454 bp BamHI-ClaI fragment from pUAF0-8 and the 2.9 kb HindIII-ClaI fragment from pUAF8-17 were ligated and inserted into the BamHI / HindIII sites of pUC19. The resulting plasmid was named pUAF0-17D. This plasmid contains 17% of the left end of the adenovirus genome lacking the E1 gene region.
[0075]
(Iii) Preparation of Bst1107-EcoRI fragment (21.6 kb) of adenovirus genome
The type 5 adenovirus DNA was digested with Bst1107 and EcoRI, separated by agarose gel electrophoresis, and the desired 21.6 kb fragment was recovered.
[0076]
(Iv) Preparation of EcoRI-SalI fragment (6.5 kb) of adenovirus genome
The SalI site of pX2S (I. Saito et. al., J. of Virology, vol. 54, p711-719, 1985) was converted to a SwaI site using a SwaI linker (manufacturer name) to obtain pX2W. After digesting pX2W with EcoRI and SwaI and separating by agarose gel electrophoresis, the desired 6.5 kb fragment was recovered.
[0077]
(V) Preparation of Shalomid (chdRBR7-11)
To remove KpnI, SmaI and BamHI from charomid 9-11 (I. Saito & G. Stark, Proc. Natl. Acad. Sci. USA, vol. 83, p8664-8668, 1986), charomid 9-11 was replaced with Asp718 and BamHI. After digestion, smoothing with Klenow enzyme, self-ligation was performed. Using this, it was transformed, and the desired shalomid was isolated and named charomid 6-11. A BamHI linker was inserted into the EcoRI site of charomid 6-11, and the resulting Shalomid was named chdRBR7-11.
[0078]
(Vi) Preparation of pAdex1c
BamHI-Bst1107 fragment (2.9 kb) of pUAF0-17D, Bst1107-EcoRI fragment (21.6 kb) of adenovirus genome and EcoRI-SwaI fragment (6.5 kb) of pX2W digested with EcoRI and Ecl36I and chdRBR7-11 Ligated. Thereafter, in vitro packaging was performed, and E. coli DH5α was infected. A transformant having the desired fragment was isolated and named pAdex1c.
[0079]
(3) Construction of cassette cosmid pAdex1cw
PAdex1c (1 μg) recovered by ethanol precipitation after cleavage with ClaI (20 units) and the following synthetic linker (1) (SEQ ID NO: 2) subjected to 5 ′ terminal phosphorylation 0.01 μg (SwaI, ClaI, SalI, Containing NruI sites) and allowed to bind overnight in a reaction (final volume 18 μl) containing ATP, T4 DNA ligase. After inactivating the ligase by incubating at 70 ° C. for 10 minutes, SwaI (20 units) was added and reacted. This cleavage was performed in order to obtain a cosmid having a structure in which only one linker was inserted by cutting out a SwaI fragment from one in which a plurality of linkers were inserted. Next, the reaction solution is subjected to Spun column (manufactured by Pharmacia) to remove a small fragment derived from the linker, and then bound overnight in a reaction solution containing ATP and T4 DNA ligase (final volume 18 μl), followed by circularization by self-annealing. went. The ligase was heat inactivated by incubating at 70 ° C. for 10 minutes and 1 μl was used for in vitro packaging. Next, the structure of cosmid DNA prepared from each colony was confirmed by simultaneous digestion with BamHI and NruI. When inserted in the target direction, a fragment of 483 bp is generated, and when inserted in the reverse direction, a fragment of 464 bp is generated. By this confirmation, a target cassette cosmid pAdex1cw (FIG. 1) was obtained.
Synthetic linker

[0080]
(4) Construction of cassette cosmid pAdexlpCAw
The plasmid pCMwCH31 constructed in {circle around (1)} was co-digested with HindIII and ClaI, blunted with Klenow enzyme, and subjected to a ligase reaction with a PmeI linker subjected to 5 ′ end phosphorylation. After inactivating the ligase by incubating at 70 ° C. for 10 minutes, Psp1406I was added and allowed to react. This cleavage was performed in order to obtain a fragment having a structure in which a Psp1406I fragment was excised from the one in which a plurality of linkers were inserted, and one linker was linked to each end of the DNA fragment. Thereafter, the reaction solution was subjected to agarose gel electrophoresis, a gel containing a 2.3 kb DNA fragment was cut out, and the DNA fragment was recovered from the gel by electrophoresis. Next, after cutting pAdexlcw with ClaI and removing the resulting small fragment with Spun column (manufactured by Pharmacia), 1 μg of DNA fragment and 0.1 μg of the above 2.3 kb DNA fragment were reacted with ATP and T4 DNA ligase. Binding overnight in liquid (final volume 18 μl). After inactivating the ligase by incubating at 70 ° C. for 10 minutes, ClaI was added to ¼ volume thereof (final volume 20 μl) to cleave the cyclic cosmid generated by self-annealing. 1 μl was used for in vitro packaging.
Next, the structure of cosmid DNA prepared from each colony was confirmed by simultaneous digestion with BamHI and XhoI. When inserted in the target direction, 483 bp and 4.8 kb are generated, and when inserted in the reverse direction, 2.7 and 2.5 kb fragments are generated. By this confirmation, the target cassette cosmid pAdexlpCAw was obtained.
[0081]
(5) Construction of cassette cosmid pAdexlCAwt (Cell engineering, Vol.13, No.8, P759)
PAdexlpCAw (1 μg) recovered by ethanol precipitation after cutting with SwaI (20 units) and the following synthetic linker (2) (SEQ ID NO: 3) (0.01 μg) (ClaI, XbaI, SpeI, PacI, SwaI, and ClaI sites) were mixed and allowed to bind overnight in a reaction solution (final volume 18 μl) containing ATP, T4 DNA ligase. After inactivating the ligase by incubating at 70 ° C. for 10 minutes, PacI (20 units) was added and reacted. This cleavage was carried out in order to obtain a cosmid having a structure in which only one linker was inserted by cutting out a PacI fragment from one in which a plurality of linkers were inserted. Next, the reaction solution is subjected to Spun column (manufactured by Pharmacia) to remove a small fragment derived from the linker, and then bound overnight in a reaction solution containing ATP and T4 DNA ligase (final volume 18 μl), and circularization by self-annealing is performed. went. 1 μl of heat inactivated ligase by incubating at 70 ° C. for 10 minutes was used for in vitro packaging. Next, the structure of cosmid DNA prepared from each colony was confirmed by simultaneous digestion with XbaI and XhoI. When inserted in the target direction, a fragment of 552 bp is generated, and when inserted in the reverse direction, a fragment of 568 bp is generated. By confirming this, the target cassette cosmid pAdexlCAwt (FIG. 2) was obtained.
Synthetic linker structure

[0082]
Example 2 (construction of loxP insertion cosmid)
(1) Preparation of pA60X99X
After cutting pAdexlCAwt (0.5 μg) in 20 μl of a reaction system containing BamHI (15 units), BamHI was inactivated by heat treatment (70 ° C., 15 minutes). Next, ATP and T4 DNA ligase were added in the ligase reaction buffer using the ¼ volume, and allowed to bind overnight in a final volume of 20 μl. Next, 10 μl of this reaction mixture was used to transform E. coli DH5α, and plasmid DNA was prepared from the resulting transformant to obtain the desired plasmid pA60X99X.
[0083]
(2) Preparation of pA60X99 (removes XbaI sites other than adenovirus) pA60X99X (5 μg) was reacted in 50 μl of a reaction system containing XbaI (10 units) for 5 minutes, and the reaction mixture was subjected to agarose gel electrophoresis. A gel containing a 23.8 kb DNA fragment cleaved at only one of the XbaI sites was excised, and the DNA fragment was recovered from the gel by electrophoresis. Next, 0.2 μg of this fragment was reacted in 50 μl of a reaction system containing 5 units of Klenow enzyme (Takara Shuzo Co., Ltd.) to smooth both ends. Further, a reaction solution containing 1/5 of this amount, ATP and T4 DNA ligase (final) Binding overnight in a volume of 20 μl). Next, 10 μl of this reaction mixture was used to transform E. coli DH5α, and plasmid DNA was prepared from the resulting transformant. These plasmid DNAs were co-digested with BsrGI and XbaI, resulting in a 6.2 kb fragment, ie, plasmid pA60X99 (FIG. 3) having the structure of FIG.
[0084]
(3) Construction of pA2L60X99 (insertion of loxP into BsrGI site)
After cutting pULL2r (30 μg) in 125 μl of a reaction system containing Xhol (150 units), XhoI was inactivated by heat treatment (70 ° C., 15 minutes). Subsequently, both ends were blunted in a reaction system containing 12 units of Klenow enzyme (Takara Shuzo), followed by phenol: chloroform (1: 1) treatment and ethanol precipitation. The precipitate was obtained by centrifugation and dissolved in 60 μl of a solution (TE) in which 1 mM EDTA was added to 10 mM Tris-hydrochloric acid (pH 7.5). Next, it was allowed to bind overnight in a ligase reaction solution (final volume 50 μl) containing 1/2 of this amount and 0.2 μg of 5′-terminal phosphorylated KpnI linker (Takara Shuzo), ATP and T4 DNA ligase. Next, the ligase was inactivated by heat treatment (70 ° C., 15 minutes), and then digested in 80 μl of a reaction system containing Asp718 (100 units). The reaction mixture was subjected to agarose gel electrophoresis, a gel containing a 64 bp DNA fragment containing loxP was cut out, and the DNA fragment was recovered from the gel by electrophoresis.
[0085]
In addition, said pULL2r was prepared as follows. pUC119 (manufactured by Takara Shuzo) was cleaved with the restriction enzyme Ecl136II, treated with alkaline phosphatase, and then the MloI site and the XhoI site at the ends, which were combined with each other and contained a loxP sequence designed to generate an NruI site. A ligation reaction with a synthetic DNA fragment (SEQ ID NO: 4) was performed to obtain a plasmid pULL2r in which two of the synthetic DNA fragments were inserted.

(The underlined sequence is the loxP site.)
[0086]
On the other hand, plasmid pA60X99 (10 μg) was cleaved in 50 μl of a reaction system containing BsrGI (50 units), the reaction mixture was subjected to agarose gel electrophoresis, a gel containing a 23.8 kb DNA fragment was excised, and DNA was extracted from the gel by electrophoresis. Fragments were collected. This DNA fragment was bound overnight in a ligase reaction solution (final volume of 25 μl) containing 0.005 μg of the 64 bp DNA fragment containing loxP and ATP and T4 DNA ligase. Sterile water and BsrGI reaction buffer were added to 1/2 volume of this to make 18 μl, and the ligase was heat-inactivated by incubating at 70 ° C. for 10 minutes. Further, 20 units of BsrGI was added (final volume 20 μl) and reacted at 37 ° C. for 1 hour to cleave the cyclic pA60X99 produced by self-annealing. Next, 10 μl of this reaction mixture was used to transform E. coli DH5α, and plasmid DNA was prepared from the resulting transformant.
[0087]
In order to confirm the direction of the inserted loxP, ApaI and MluI were simultaneously digested, and the reaction mixture was subjected to agarose gel electrophoresis. When inserted in the intended direction, 366 and 219 bp fragments are generated, and when inserted in the reverse direction, 334 and 251 bp fragments are generated. In addition, when digested with NruI, a fragment of 573 bp is generated when inserted in the intended direction, and a fragment of 533 bp is generated when inserted in the reverse direction. Furthermore, when co-digested with DraI and KpnI, a fragment of 384 bp is generated when one loxP is inserted in the target direction and 320 bp is inserted when two are inserted. The target plasmid pA2L60X99 (FIG. 4) was obtained that satisfied all these three conditions, that is, one loxP inserted in the target direction.
[0088]
(4) Construction of pA2L3L6099 (insertion of loxP into XbaI site)
After cutting pULL2r (20 μg) in 100 μl of a reaction system containing XhoI (100 units), XhoI was inactivated by heat treatment (70 ° C., 15 minutes). Subsequently, both ends were blunted in a reaction system containing 8 units of Klenow enzyme (Takara Shuzo), followed by phenol: chloroform (1: 1) treatment, followed by ethanol precipitation. The precipitate was obtained by centrifugation and dissolved in 30 μl of TE. Ligase was bound overnight in a reaction solution (final volume of 50 μl) containing 0.4 μg of 5′-terminal phosphorylated SpeI linker (Takara Shuzo), ATP and T4 DNA ligase and incubated at 70 ° C. for 10 minutes. Heat inactivated. Furthermore, after digesting with SpeI (54 units), the reaction mixture was subjected to agarose gel electrophoresis, a gel containing a 64 bp DNA fragment containing loxP was excised, and the DNA fragment was recovered from the gel by electrophoresis.
[0089]
On the other hand, pA2L60X99 (10 μg) was cleaved in 50 μl of a reaction system containing XbaI (100 units), the reaction mixture was subjected to agarose gel electrophoresis, a gel containing a 23.8 kb DNA fragment was excised, and DNA was extracted from the gel by electrophoresis. It was collected. The DNA fragment was bound overnight in a reaction solution (final volume 16 μl) containing 0.005 μg of the 64 bp DNA fragment containing loxP and ATP and T4 DNA ligase. To this, 14 μl of TE diluted 5 times was added, and the ligase was heat-inactivated by incubating at 70 ° C. for 10 minutes. Next, 20 units of XbaI was added to a quarter of this amount (final volume 20 μl), and the cyclic pA2L60X99 produced by self-annealing was cleaved. Escherichia coli DH5α was transformed with 10 μl of this reaction mixture, and plasmid DNA was prepared from the resulting transformant.
[0090]
In order to confirm the direction of the inserted loxP, BglII and MluI were simultaneously digested, and the reaction mixture was subjected to agarose gel electrophoresis. When inserted in the intended direction, 366 and 503 bp fragments are generated, and when inserted in the reverse direction, 398 and 471 bp fragments are generated. When ApaI and MluI are digested simultaneously, a fragment of 660 bp is generated when inserted in the target direction, and a fragment of 628 bp is generated when inserted in the reverse direction. When digested with EcoNI and MluI, a fragment of 311 bp is generated when inserted in the target direction and 343 bp when inserted in the reverse direction. Further, when digested simultaneously with HpaI and SacI, a fragment of 397 bp is generated when one loxP is inserted in the target direction, and a 461 bp fragment is generated when two are inserted. The objective plasmid pA2L3L6099 (FIG. 5) in which all these four conditions were satisfied, ie, one loxP was inserted in the intended direction was obtained.
[0091]
(5) Production of pAdex2L3LCAwt
pAdex1CAwt (10 μg) was cleaved in 100 μl of a reaction system containing Csp45I (40 units), and then BamHI (30 units) and EcoRI (40 units) were sequentially added to the reaction solution. A gel containing a 21 kb DNA fragment was excised and the DNA fragment was recovered from the gel by electrophoresis. The cleavage with Csp451 and EcoRI is for preventing contamination of other fragments when recovering the 21 kb BamHI DNA fragment.
[0092]
pA2L3L6099 (5 μg) was cleaved in 50 μl of a reaction system containing BamHI (30 units), treated with phenol: chloroform (1: 1), and the aqueous layer was subjected to gel filtration with Sephadex G25 equilibrated with TE. The recovered DNA fragment was bound overnight in a reaction solution (final volume of 18 μl) containing 0.5 μg of the 21 kb DNA fragment, ATP and T4 DNA ligase described above. After heat inactivation of the ligase by incubating at 70 ° C. for 10 minutes, 1 μl was used for in vitro packaging.
[0093]
That is, Gigaback XL (manufactured by Stratagene), which is a lambda in vitro packaging kit, was used at ¼ scale, and the rest was frozen at −80 ° C. Since GIGABACK XL has a low packaging efficiency for cosmids of 42 kb or less, it can be selected to some extent from cosmids that have grown with inserts. In this experiment, when 10 colonies were picked up, most of them contained an insert, and the target clone (a clone in which the virus genome was correctly linked) could be easily obtained. The handling of the cosmid was carried out according to a conventional method (Izumi Saito et al., Experimental Medicine: 7: 183-187, 1989).
[0094]
The packaged cosmid was infected into E. coli DH5α (GibcoBRL). That is, three Ap⁺(Ampicillin added) Agar plate and 5 ml of Ap⁺LB (pool) was inoculated with 1/200 volume, 1/20 volume, 1/2 volume and the remaining total volume, respectively, and cultured overnight.
Pool miniprep DNA was extracted and prepared, and the ratio of inserts inserted by digestion with restriction enzyme DraI was examined. Take the whole colony with agar and add 1.5ml of Ap⁺Miniprep DNA was prepared by overnight culture in LB. Next, the structure of cosmid DNA prepared from each colony was confirmed by DraI digestion. When inserted in the target direction, 891 bp is generated, and when inserted in the reverse direction, a 1.4 kb fragment is generated. As a result, the target cassette cosmid pAdex2L3LCAwt was obtained.
[0095]
Example 3
(Preparation of adenovirus DNA-terminal protein complex (Ad5 dlX DNA-TPC and Adex1CANLacZ DNA-TPC))
(1) As adenovirus DNA, Ad5 dlX (I. Saito et al., J. Virology, vol. 54, 711-719 (1985)) or Adex1CANLacZ is used. Ad5 dlX was infected to HeLa cells (for 10 Roux) and dex1CANLacZ was infected to 293 cells, respectively, and cultured.
That is, a virus solution of Ad5-dlX or Adex1CANLacZ (-10⁹PFU / ml) was infected with 0.2 ml / Roux, and after 3 days, detached cells were collected by centrifugation. Most of the adenovirus particles are in the cell nucleus, not in the medium, which has the advantage that the virus can be purified from infected cells. (The following operations are performed aseptically.)
[0096]
(2) The obtained cells were suspended in Tris-HCl (pH 8.0), the cells were disrupted using a sealed sonicator, and the virus was released from the cells.
(3) After removing the precipitate from the resulting crushed material by centrifugation, put the cesium chloride solution (specific gravity 1.43) into the ultracentrifuge SW28 tube, overlay the supernatant on it, and concentrate by cushion centrifugation Went.
[0097]
(4) The virus layer just below the interface was transferred to a SW50.1 tube. The virus layer directly under the interface was usually visible, and 5 ml of the virus layer and cesium chloride under the virus layer were collected. At the same time, the other was filled with a cesium chloride solution (specific gravity 1.34).
These were ultracentrifuged overnight at 35 krpm and 4 ° C. Next, the white virus band was separated and transferred to a tube with a gradient. Further, ultracentrifugation was performed at 35 krpm and 4 ° C. for 4 hours or more.
[0098]
(5) A white virus band was separated and mixed with an equal amount of 8M guanidine hydrochloride at room temperature, and 4M guanidine hydrochloride saturated cesium chloride was added to fill the VTi65 tube. The particle protein was denatured and dissociated by 4M guanidine hydrochloride, and DNA-TPC was released.
[0099]
(6) The above tube was ultracentrifuged at 55 krpm and 15 ° C. overnight, fractionated by 0.2 ml, 1 μl of each was mixed with 20 μl of 1 μg / ml ethidium bromide aqueous solution, and the DNA was stained by fluorescence staining. Check for presence. A few fractions containing DNA were collected.
(7) Dialyzed overnight (twice) in 500 ml of TE and stored at -80 ° C. The amount of Ad5dlX and dex1CANLacZ DNA-TPC obtained in this way was OD₂₆₀Was calculated in the same manner as normal DNA.
(8) The obtained Ad5dlX and Adex1CANLacZ DNA-TPC were cleaved with a sufficient amount of AgeI for 2 hours for preparation of the recombinant adenovirus in the third step, gel-filtered on a Sephadex G25 column, and stored at −80 ° C. .
[0100]
DNA-TPC can be cleaved with restriction enzymes, dialyzed, and gel filtered, but cannot be subjected to electrophoresis, phenol treatment, or ethanol precipitation. Since the concentration method was only cesium chloride equilibrium centrifugation, it was kept as thick as possible. About 300 μg of DNA-TPC could be obtained from 10 Roux infected cells.
{Circle around (9)} A portion was collected, 10 μl of BPB buffer for electrophoresis was added, 1 μl of proteinase K (10 mg / ml) was added, and the mixture was reacted at 37 ° C. for 10 minutes to digest the terminal protein. Phenol was extracted and the supernatant was separated by agarose gel electrophoresis to confirm complete cleavage.
The restriction enzyme buffer in AgeI-cut DNA-TPC was removed by centrifugal gel filtration, and then dispensed and stored at -80 ° C.
[0101]
Example 4
(Production of loxP-inserted recombinant adenovirus (Ad5dlX2L3L or Adex2L3LCANLacZ))
NLacZ is obtained by adding a nuclear translocation signal of SV40 to the N-terminus of the E. coli LacZ gene.
(1) One 6 cm and 10 cm petri dishes of 293 cells cultured in DME supplemented with 10% FCS were prepared.
(2) 8 μg of cosmid pAdex2L3LCAwt DNA inserted with loxP incorporating an expression unit and 1 μg of Ad5dlX DNA-TPC or Age1 digested with Age5dlX DNA-TPC were mixed with Cellfect (Pharmacia) kit. The transfection was performed on one 6 cm petri dish by the calcium phosphate method. The mixed solution was dropped from above the medium of 6 cm petri dish and the culture was continued.
Incubate overnight (about 16 hours), change the culture medium in the morning, and in the evening, add 3% collagen-coated 96-wells (stock solution, 10-fold dilution, 100-fold dilution) to each well using 5% FCS-added DME. Re-rolled with 0.1 ml per unit. In order to prevent the number of cells from greatly differing between each plate, 293 cells of a 10 cm dish were mixed and spread on two dilutions.
[0102]
(3) After 3 to 4 days and 8 to 10 days, 50 μl of 10% FCS-added DME was added to each well. When 293 cells became thin, it was added early.
Wells in which the virus had grown and the cells had died appeared between 7 and 20 days. Each time the cells in the well were completely killed, the culture solution (every dead cells) was aseptically transferred to a sterilized 1.5 ml tube with a sterile Pasteur pipette, snap frozen with dry ice and stored at −80 ° C.
(4) The determination was completed in 15-25 days. Select about 10 culture tubes collected from wells where cells died relatively late, crush ultrasonically and centrifuge at 5000 rpm for 10 minutes, and use the supernatant obtained as the first seed solution at -80 ° C. saved.
This is because a well in which virus propagation has occurred earlier is likely to be a mixed infection of a plurality of virus strains.
[0103]
(5) Prepare 293 cells in a 24-well plate, and add 2% each of 5% FCS-DME (0.4 ml / well) and 10 μl of the primary virus solution.
(6) When the cells are completely killed in about 3 days, a supernatant is obtained by sonication and centrifugation in the same manner as in the preparation of the primary virus solution, and this is used as a secondary virus solution (second seed) at -80. Stored at ° C. The other 1-well dead cells were centrifuged at 5000 rpm for 5 minutes, the supernatant was discarded, and only the cells were stored at −80 ° C. (cell pack). When cell packs of 10 virus strains were collected, the total DNA of infected cells was extracted by the following method. To the cell pack, 400 μl of TNE for cell DNA (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA), 4 μl of proteinase K (10 mg / ml) and 4 μl of 10% SDS were added.
[0104]
(7) After treatment at 50 ° C. for 1 hour, the nucleic acid obtained by phenol / chloroform extraction twice, chloroform extraction twice, and ethanol precipitation was dissolved in 50 μl TE containing 20 μg / ml RNase.
15 μl of the expression unit was cleaved with XhoI, an enzyme containing CG as a recognition sequence among the enzymes that cleave the expression unit, and the electrophoresis was performed overnight on an agarose gel with a length of about 15 cm along with XhoI cleavage of the expression cosmid cassette. The patterns were compared. Since XhoI has a recognition site in the inserted loxP sequence, a clone showing a cleavage pattern in which two loxPs are inserted is selected. Clones that seemed unexplainable bands were discarded because they could be mixed with deleted viruses.
[0105]
Example 5
(Production and structure confirmation of E2A gene-deficient adenovirus)
Recombinant adenoviruses Adex2L3LCANLacZ and Adex1CANCre were infected with 293 cells at moi = 10 and 3, respectively, and cultured. NCre is obtained by adding SV40 nuclear translocation signal to the N-terminal of Cre, like NLacZ.
On day 4, cells were collected and DNA was prepared by the method described above. Generation of dex123CANLacZ having a structure in which a region sandwiched by loxP (including the E2A gene) was excised was confirmed by two methods.
[0106]
1. SmaI digestion
As a result of gel electrophoresis after SmaI digestion, a 4.7 kb fragment produced by cutting out the region sandwiched by loxP was observed. A comparison of the density of this band and the 4.45 kb band commonly found in dex2L3LCANLacZ, dex1CANCre, and dexd123CANLacZ revealed that about 30% of the recovered recombinant adenovirus was dex123CANLacZ.
[0107]
2. Confirmation by PCR
PCR was performed under the following conditions using 0.1 ng of the prepared DNA as a template, and the product was analyzed by agarose gel electrophoresis. The primers used were the following oligonucleotide (1) (SEQ ID NO: 5), oligonucleotide (2) (SEQ ID NO: 6), oligonucleotide (3) (SEQ ID NO: 7) and oligonucleotide (4) ( SEQ ID NO: 8).

PCR reaction solution composition (total volume 20 μl)
Tris.HCl (pH 8.3) 10 mM
KCl 50 mM
MgCl₂                            1.5 mM
dNTP mixture 0.2 mM
Each primer 0.2 μM
Template DNA 0.1ng
Taq polymerase 0.5unit
PCR reaction conditions
Double-strand dissociation: 95 ° C for 1.5 minutes
Annealing: 64 ° C 1.0 min
Elongation reaction: 70 ° C 1.0 min
Reaction cycle: 30 times
The result is shown in FIG.
When (1) and (4) were used as primers, a band estimated to be 393 bp was detected, revealing the presence of dex123CANLacZ from which the E2A gene was deleted (FIG. 6, lane 1).
When (2) and (3) were used, a band estimated to be 221 bp was detected, confirming the presence of the circular E2A gene excised by the Cre gene product (FIG. 6, lane 2).
As described above, the results of 1 and 2 revealed the generation of dex123CANLacZ from which the region sandwiched by loxP (including the E2A gene) was removed from dex2L3LCANLacZ.
[0108]
Reference example 1
<Preparation of recombinant adenovirus vector having recombinase Cre gene and CAG promoter>
(1) Preparation of cassette cosmid for recombinase Cre gene expression
(1) Escherichia coli phage P1 DNA (ATCC 11303-B23) containing the recombinase Cre gene is used as a template, and the following oligonucleotide (SEQ ID NO: 9) is used as a 5′-primer and the following (SEQ ID NO: 10 is used as a 3′-primer. ) As a heat-resistant polymerase, manufactured by NEB Vent^RThe product was subjected to agarose gel electrophoresis under the following conditions, and a band of about 1 kb was cut out to obtain a DNA fragment of about 1 kb containing the recombinase Cre gene.

(The underlined portion is a restriction enzyme recognition site.)
[0109]
PCR reaction conditions
Buffer: 10 mM KCl, 20 mM Tris-HCl (pH 8.8), 10 mM (NH_Four)₂SO_Four2 mM MgSO_Four0.1% Triton X-100 (using buffer attached to NEB)
Thermostable polymerase: 2 units
dNTP: 400 μM
Primer: 1 μM
P1 phage DNA: 1 ng
Double-strand dissociation temperature: 95 ° C for 1.5 minutes
Annealing temperature: 60 ° C for 1.5 minutes
Elongation reaction temperature: 74 ° C for 2.0 minutes
Reaction cycle: 20 times
[0110]
This fragment and pUC19 (Takara Shuzo) were simultaneously digested with restriction enzymes PstI (Takara Shuzo) and XbaI (Takara Shuzo), respectively, recovered, mixed to a molar ratio of about 3: 1, and T4 DNA ligase (Takara Shuzo). Ligation reaction was carried out. Further, E. coli strain JM109 (ATCC 53323) was transformed with this reaction mixture. A transformant was picked up from an LB agar plate supplemented with ampicillin (100 μg / ml) to obtain a plasmid pUCCre containing the recombinase Cre gene.
[0111]
Next, 1 μg of the cassette cosmid pAdex1CAwt containing CAG promoter cut with SwaI and pUCCre were digested simultaneously with PstI and XbaI, and further blunted at both ends with Klenow enzyme (Takara Shuzo). 1 μg was mixed.
[0112]
(2) Next, ethanol was added to the mixed solution to precipitate a cosmid. The precipitate was obtained by centrifugation and dissolved in a 5-fold dilution of a solution (TE) in which 1 mM EDTA was added to 10 mM Tris-hydrochloric acid (pH 7.5).
(3) ATP and T4 DNA ligase was added to the obtained cosmid in a ligase reaction buffer and allowed to bind overnight in a final volume of 7 μl. Subsequently, sterilized water and SwaI reaction buffer were added to make 48 μl, and then the ligase was heat-inactivated at 70 ° C. for 10 minutes.
At this time, unlike the plasmid, the cosmid efficiently packages macromolecules bonded to a linear tandem rather than a ring.
[0113]
(4) 2 μl of SwaI (manufactured by Boehringer) was added and cut at 25 ° C. for 1 hour.
The meaning of cleaving SwaI is that when the cassette cosmid recombines without adding the expression unit, the Swal recognition sequence is regenerated, so in this step, the cosmid that does not incorporate the expression unit is recut to prevent colony formation Because. This method is a powerful way to select only cassette cosmids with inserts.
(5) According to a conventional method (Molecular Cloning vol.3 E.34), phenol extraction of the cassette cosmid, centrifugation, and gel filtration were performed.
(6) Swal cutting was performed again. That is, 5 μl of SwaI was added to the SwaI reaction buffer and cleaved at 25 ° C. for 2 hours. The reason is as described above.
[0114]
(7) In vitro packaging was performed on 1 μl of the obtained cosmid.
That is, Gigaback XL (manufactured by Stratagene), which is a lambda in vitro packaging kit, was used at ¼ scale, and the rest was frozen at −80 ° C. Since GIGABACK XL has a low packaging efficiency for cosmids of 42 kb or less, it can be selected to some extent from cosmids that have grown with inserts. In this experiment, when 10 colonies were picked up, most of them contained inserts, and clones with the desired orientation (leftward) could be easily obtained.
The handling of the cosmid was carried out according to a conventional method (Izumi Saito et al., Experimental Medicine: 7: 183-187, 1989).
[0115]
(8) The packaged cosmid was infected with E. coli DH1 (ATCC 33849).
That is, three Ap⁺(Ampicillin added) Agar plate and 5 ml of Ap⁺LB (pool) was inoculated with 1/200 volume, 1/20 volume, 1/2 volume and the remaining total volume, respectively, and cultured overnight.
Pool miniprep DNA was extracted and prepared, and the ratio of the insert containing the whole enzyme was examined. Take the whole colony with agar and add 1.5ml of Ap⁺Miniprep DNA was prepared by overnight culture in LB.
(9) Next, the orientation and structure of the expression unit were confirmed by restriction enzyme digestion.
A plasmid containing an expression unit but lacking most of adenovirus DNA was prepared using NruI and ligase, DNA was prepared, and final confirmation of cDNA cloning was performed.
[0116]
(2) Preparation of adenovirus DNA-terminal protein complex (Ad5 dlX DNA-TPC)
(1) Ad5 dlX (I. Saito et al., J. Virology, vol. 54, 711-719 (1985)) was used as adenovirus DNA. Ad5 dlX was infected to 293 cells (for 10 Roux) and cultured.
That is, Ad5-dlX virus solution (-10⁹PFU / ml) was infected with 0.2 ml / Roux, and after 3 days, the detached cells were collected by centrifugation at 1500 rpm for 5 minutes. Most of the adenovirus particles are in the cell nucleus, not in the medium, which has the advantage that the virus can be purified from infected cells. (The following operations were performed non-aseptically.)
[0117]
(2) The obtained cells are suspended in 20 ml of 10 mM Tris-HCl (pH 8.0), and the cells are crushed at 200 W for 2 minutes (30 seconds × 4) using a sealed sonicator, and the virus is intracellularized. Released from.
In order to release the virus from the cell, 5 ml or less may be used for freezing and thawing, but a sonicator is convenient for a volume larger than that. However, be sure to use a sealed type (with a special cup). The normal throw type is dangerous even in a safety cabinet.
[0118]
(3) After removing the precipitate by centrifugation (10 krpm, 10 minutes), 15 ml of cesium chloride solution (specific gravity 1.43) was placed in an ultracentrifuge SW28 tube, and the supernatant was placed on it. The layers were layered and concentrated by cushion centrifugation (25 krpm, 1 hour, 4 ° C.).
(4) The virus layer just below the interface was transferred to a SW50.1 tube. The virus layer directly under the interface was usually visible, and 5 ml of the virus layer and cesium chloride under the virus layer were collected. At the same time, the other was filled with a cesium chloride solution (specific gravity 1.34).
These were ultracentrifuged overnight at 35 krpm and 4 ° C. Next, the white virus band was separated and transferred to a tube with a gradient. Further, ultracentrifugation was performed at 35 krpm and 4 ° C. for 4 hours or more.
[0119]
(5) A white virus band was separated and mixed with an equal amount of 8M guanidine hydrochloride at room temperature, and 4M guanidine hydrochloride saturated cesium chloride was added to fill the VTi65 tube. The particle protein was denatured and dissociated by 4M guanidine hydrochloride, and DNA-TPC was released. Ethidium bromide was not available because a method for later removal was not established.
[0120]
(6) The above tube was ultracentrifuged at 55 krpm and 15 ° C. overnight, fractionated by 0.2 ml, 1 μl of each was mixed with 20 μl of 1 μg / ml ethidium bromide aqueous solution, and the DNA was stained by fluorescence staining. The presence or absence was confirmed. A few fractions containing DNA were collected.
(7) Dialyzed overnight (twice) in 500 ml of TE and stored at -80 ° C. The amount of Ad5dlX DNA-TPC thus obtained was OD₂₆₀Was calculated in the same manner as normal DNA.
[0121]
(8) The obtained Ad5d1X DNA-TPC was cleaved with a sufficient amount of EcoT22I for 2 hours for preparation of the recombinant adenovirus in the third step, and then stored at −80 ° C.
[0122]
DNA-TPC could be digested with restriction enzymes, dialyzed, and gel filtered, but could not be electrophoresed, treated with phenol, or precipitated with ethanol. Since the concentration method was only cesium chloride equilibrium centrifugation, it was kept as thick as possible. About 300 μg of DNA-TPC could be obtained from 10 Roux infected cells.
{Circle around (9)} A portion was collected, 10 μl of BPB buffer for electrophoresis was added, 1 μl of proteinase K (10 mg / ml) was added, and the mixture was reacted at 37 ° C. for 10 minutes to digest the terminal protein. Phenol was extracted and the supernatant was separated by agarose gel electrophoresis to confirm complete cleavage.
The restriction enzyme buffer in EcoT22I-cleaved DNA-TPC was removed by centrifugal gel filtration, and then dispensed and stored at -80 ° C.
[0123]
(3) Isolation of recombinant virus and preparation of high titer virus solution
(1) One 6 cm and 10 cm petri dishes of 293 cells cultured in DME supplemented with 10% FCS were prepared.
(2) 8 μg (preferably 3 to 9 μg) of pAdex1w DNA incorporating the expression unit and 1 μg of Ad5dlX DNA-TPC cleaved with EcoT22I were mixed, and one 6 cm petri dish using a Cellfect (Pharmacia) kit. The cells were transfected by the calcium phosphate method. The mixed solution was dropped from above the medium of 6 cm petri dish and the culture was continued.
Incubate overnight (about 16 hours), change the culture medium in the morning, and in the evening, add 3% collagen-coated 96-wells (stock solution, 10-fold dilution, 100-fold dilution) to each well using 5% FCS-added DME. Re-rolled with 0.1 ml per unit. In order to prevent the number of cells from greatly differing between each plate, 293 cells of a 10 cm dish were mixed and spread on two dilutions.
[0124]
(3) After 3 to 4 days and 8 to 10 days, 50 μl of 5% FCS-added DME was added to each well. When 293 cells became thin, it was added early.
Wells with virus growth and cell death appeared between 7 and 15 days. Each time the cells in the well were completely killed, the culture solution (every dead cells) was aseptically transferred to a sterilized 1.5 ml tube with a sterile Pasteur pipette, snap frozen with dry ice and stored at −80 ° C.
(4) The determination was completed in 15-18 days. Select about 10 culture tubes collected from wells where cells died relatively slowly, freeze and thaw 6 times, then centrifuge at 5 krpm for 10 minutes and store the supernatant at -80 ° C. as the first virus solution (first seed) did.
This is because a well in which virus propagation has occurred earlier is likely to be a mixed infection of a plurality of virus strains.
[0125]
(5) Prepare 293 cells in a 24-well plate, and add 2% each of 5% FCS-DME (0.4 ml / well) and 10 μl of the primary virus solution.
(6) When the cells are completely killed in about 3 days, 1 well is obtained by freezing and thawing and centrifuging 6 times in the same manner as in the preparation of the primary virus solution. Stored at 80 ° C. The titer of the secondary virus solution is 10⁷-10⁸It was about PFU / ml. The other 1-well dead cells were centrifuged at 5 krpm for 5 minutes, the supernatant was discarded, and only the cells were stored at −80 ° C. (cell pack). When cell packs of 10 virus strains were collected, the total DNA of infected cells was extracted by the following method. To the cell pack, 400 μl of TNE for cell DNA (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA), 4 μl of proteinase K (10 mg / ml) and 4 μl of 10% SDS were added.
[0126]
(7) After treatment at 50 ° C. for 1 hour, the nucleic acid obtained by phenol / chloroform extraction twice, chloroform extraction twice, and ethanol precipitation was dissolved in 50 μl TE containing 20 μg / ml RNase.
15 μl of the expression unit was cleaved with XhoI, an enzyme containing CG as a recognition sequence among the enzymes that cleave the expression unit, and the electrophoresis was performed overnight on an agarose gel with a length of about 15 cm along with XhoI cleavage of the expression cosmid cassette. The patterns were compared. A band in which a band from the break point in the expression unit to the left end of the adenovirus genome appeared correctly was selected. In addition, clones in which unexplainable bands appear thin were discarded because they could be mixed with viruses with deletions.
Since adenoviral DNA grows to 10,000 copies per cell, the total DNA can be extracted together with the cellular DNA, and viral DNA bands can be seen by restriction enzyme digestion. Enzymes containing CG in the recognition sequence, such as Xhol, do not cleave cellular DNA, so the pattern is easy to see. When using other enzymes, it was necessary to keep uninfected 293 cell DNA in control. (A band derived from a repetitive sequence of human cells appeared).
[0127]
(8) Collagen-coated 150 cm of 0.1 ml of the secondary virus solution of the target virus strain identified by Xhol cleavage²293 cells in a bottle (25 ml medium) were infected.
When the cells died after 3 days, 25 ml of medium with dead cells was aseptically disrupted in a sealed sonicator 200w with a maximum output of 2 minutes (30 seconds × 4 times) to release the virus.
The mixture was centrifuged at 3 krpm and 4 ° C. for 10 minutes to remove the precipitate, dispensed into 13 ml each of 2 ml in a 5 ml freezing tube, rapidly frozen with dry ice and stored at −80 ° C. to prepare a tertiary virus solution. The tertiary virus solution is a solution containing the recombinant adenovirus of the present invention.⁹It had a high titer of about PFU / ml.
In addition, 5 μl of the tertiary virus solution was infected to one well of 293 cells in a 24-well plate, and the enzyme cleavage pattern of the grown viral DNA was confirmed by the above method. If it is suspected that it is a deletion virus or a mixture with the parent virus, the deletion virus that was already slightly mixed at the secondary virus solution stage may have appeared due to its rapid growth, All the tertiary seeds were discarded, and the target virus was purified from another primary virus solution by redoing again or by limiting dilution from the primary virus solution.
[0128]
【The invention's effect】
According to the present invention, a recombinant DNA virus capable of stably introducing a foreign gene into a wide range of animal cells can be provided. The present invention also provides a simple method for producing this recombinant DNA virus. In particular, the recombinant adenovirus of the present invention is useful for the treatment of genetic diseases.
[0129]
[Sequence Listing]

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[0140]

[Brief description of the drawings]
FIG. 1 is a conceptual diagram showing the structure of a cosmid pAdex1cw.
FIG. 2 is a conceptual diagram showing the structure of cosmid pAdex1CAwt.
FIG. 3 is a conceptual diagram showing the structure of plasmid pA60X99.
FIG. 4 is a conceptual diagram showing the structure of plasmid pA2L60X99.
FIG. 5 is a conceptual diagram showing the structure of plasmid pA2L3L6099.
FIG. 6 is a diagram showing the results of performing PCR using DNA extracted from cells collected after co-infection of 293 cells with recombinant adenoviruses Adex2L3LCANLacZ and Adex1CANCre, and using this as a template. Lane 1 shows an electropherogram when primers (1) and (4) are used, and lane 2 shows an electropherogram when primers (2) and (3) are used.

Claims (1)

A recombinant adenovirus for infection of animal cells, wherein a recombinase recognition sequence is inserted between the stop codon of the E2A gene and the stop codon of the L3 gene.

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